One possible presynaptic effector for ethanol is the Munc13-1 protein. Herein, we show that ethanol binding to the rat Munc13-1 C1 domain, at concentrations consistent with binge exposure, reduces diacylglycerol (DAG) binding. The inhibition of DAG binding is predicted to reduce the activity of Munc13-1 and presynaptic release. In Drosophila, we show that sedating concentrations of ethanol significantly reduce synaptic vesicle release in olfactory sensory neurons (OSNs), while having no significant impact on membrane depolarization and Ca2+ influx into the presynaptic compartment. These data indicate that ethanol targets the active zone in reducing synaptic vesicle exocytosis. Drosophila, haploinsufficent for the Munc13-1 ortholog Dunc13, are more resistant to the effect of ethanol on presynaptic inhibition. Genetically reducing the activity of Dunc13 through mutation or expression of RNAi transgenes also leads to a significant resistance to the sedative effects of ethanol. The neuronal expression of Munc13-1 in heterozygotes for a Dunc13 loss-of-function mutation can largely rescue the ethanol sedation resistance phenotype, indicating a conservation of function between Munc13-1 and Dunc13 in ethanol sedation. Hence, reducing Dunc13 activity leads to naïve physiological and behavioral resistance to sedating concentrations of ethanol. We propose that reducing Dunc13 activity, genetically or pharmacologically by ethanol binding to the C1 domain of Munc13-1/Dunc13, promotes a homeostatic response that leads to ethanol tolerance.Ethanol has robust effects on presynaptic activity in many neurons, however, it is not yet clear how this drug acts within this compartment to change neural activity, nor the significance of this change on behavior and physiology  Drosophila Dunc13 ortholog leads to a homeostatic change that promotes behavioral and physiologic resistance to intoxicating levels of ethanol. Hence, Unc13 proteins are likely sites for ethanol’s action within the presynaptic compartment that bring about tolerance.At relatively low concentrations, ethanol inhibits the activity of many presynaptic termini . HomeostAddiction to alcohol remains one of the most significant mental health problems throughout of the world. A major challenge is to understand how ethanol changes behavior and the brain during the descent into addiction. A promising approach is to examine endophenotypes for this disease in model systems . One majCaenorhabditis elegans, respectively  was generously provided by the Kyoto Stock Center. KK101383Dunc13 RNAi line (RRID: FlyBase_FBst0479208) was provided by Vienna Drosophila Resource Center. The elav-Gal4 , UAS-Arclight , UAS-GCaMP5 (RRID: FlyBase_FBst0042037) and JF02440Dunc13 RNAi line (RRID: BDSC_29548) were provided by Bloomington Drosophila Stock Center. The n-syb-Gal4 was generously provided by Herman Dierick (Baylor College of Medicine). The Or42b-Gal4 was kindly provided by Scott Pletcher (University of Michigan). The tublin-Gal80ts2, 506ry, and UAS-pHluorin were generously provided by Ronald L. Davis .All flies were raised on standard cornmeal food at 25°C on a 12/12 h light/dark cycle. All the stocks were out-crossed into the Canton-S background for a minimum of six generations before behavioral analysis. The Munc13.1-EGFP cDNA was digested from the pBRETU vector , and cloP84200/+Dunc13 genotype was generated by crossing virgin females of 506ry, with w+; +; 506ry; P84200Dunc13/Dcimales. For the functional complementation of P84200Dunc13, the experimental genotype was generated by crossing virgin females of w+; tublin-Gal80ts; 506ry; Dunc13/Dci, with w+; UAS-Munc13-1 (or UAS-dunc13A); elav-Gal4;+ males. For optical imaging, the genotype Or42b-Gal4/+; UAS- pHluorin /+ was generated by crossing virgin females of Or42b-Gal4, with UAS-pHluorin  males, and the genotype Or42b-Gal4/+; UAS- pHluorin/+; P84200Dunc13/+ was generated by crossing virgin females of Or42b-Gal4;; P84200Dunc13/Dci, with UAS-pHluorin males.For the ethanol sedation assay, the n = 30 flies per vial) for 24 h before behavioral testing. They were then transferred to the empty plastic vials of the test apparatus and exposed to a stream of ethanol vapor (50% unless noted) at a flow of 250 ml/min for 1 h to test for ethanol sensitivity. The ethanol vapor was produced by bubbling fresh air through 100% ethanol and pure water and mixing the two kinds of vapors. The % ethanol refers to the percentage of the final air/ethanol stream made up of the air bubbled through ethanol. The percentage of flies sedated in each vial was recorded at 5-min intervals. A fly was counted as being sedated if it had fallen onto its back or side and could not right itself. The time to 50% LOR was calculated for each exposure tube by linear interpolation of the two time points around the median and then averaged over the number of tubes (The ethanol loss of righting reflex (LOR) assay was performed as previously described . Female of tubes .g at 4°C for 20 min, and the supernatant was collected. The ethanol concentration in the supernatant was measured using the Ethanol Assay kit . To determine whether equivalent amounts of flies were assayed for each genotype, the protein concentration in the fly extracts was measured using the DC protein assay (Bio-Rad Laboratories). Ethanol metabolism was examined by exposing flies to 50% ethanol vapor for 60 min and allowing flies to recover for a period of 0, 30, 60, or 120 min. The ethanol concentration in fly extracts was measured at the end of each recovery period.The ethanol metabolism and absorption assay has been described before . To studOptical imaging was performed on live flies as previously described with modifications . Each feBefore injection, each dissected female fly was checked through gently touching the tip of its foreleg with forceps. If the fly’s femur did not show any movement, it would be discarded since it might be dead or close to dying. To inject ethanol, a small piece of pipette tip which contained fixed fly was removed to expose its scutellum. An insect pin  was inserted between scutum and scutellum to make a hole. A glass pipette (top diameter ∼20 μm), was inserted into the hole. Then an injector  linked with the glass pipette was turned on to inject ∼0.1 μl ethanol solution into fly. The injected solution was Ringer’s solution plus 15% ethanol.After 10 min, the injected fly was imaged under an upright confocal microscope with a 20× objective . Ethyl acetate was diluted in mineral oil to 1% and delivered with an air flow at a rate of 400 ml/min bubbled through the mineral oil. The delivery of odorants was accomplished with a CS55 stimulus controller (Syntech), which added the odor to an airstream. Flies were exposed to 1-min air and then 3-s odor; there were no mechanical disturbances during the transition from air to odor.After the imaging, each fly was taken out of the pipette tip to check for viability. Most of the flies exhibited leg movements. However, if a fly’s legs showed no movement, it was considered dead, and the data were discarded.S-transferase (GST) were expressed in BL21 DE3 gold Escherichia coli (Agilent Technologies). Munc13.1C1 and its E582A mutant were purified following the methods described earlier  were incubated for 30 min at 25°C. For measuring the effect of ethanol, mixtures were incubated with ethanol (25 and 50 mM) for 30 min at 25°C. Samples were then excited at 280 nm, and emission spectra were recorded from 300 to 550 nm. Emission maxima of Munc13.1 C1 and dansyl-DAG were found to be at 337 and 500 nm, respectively. Relative FRET indices were calculated from fluorescence intensity at 500 nm using formula: [(Fi + Munc13.1C1) − ] − [(F0 + Munc13.1C1) − (F0− Munc13.1C1)], where Fi + Munc13.1C1 and Fi − Munc13.1C1 are the intensities of Dansyl-DAG in the presence and absence of Munc13.1C1, respectively, and F0+ Munc13.1C1 and F0− Munc13.1C1 are fluorescence intensities of the buffer solution in the presence or absence of Munc13.1C1, respectively. The change in fluorescence intensities for each concentration of alcohol was normalized using the equation: (1-F/F0) × 100, where F and F0 are intensities of dansyl-DAG plus Munc13.1C1 in the presence or in the absence of alcohol.The Munc13-1 C1 domain in pGEX-KG vector was kindly provided by Dr. Josep Rizo . Site-specific mutations in the Munc 13-1 C1 domain were introduced with PCR by presenting the mutation in oligonucleotide primers using the he QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies). The Munc 13-1 C1 domain wild-type or the mutant fused with glutathione  earlier . Fluores earlier . Samplest tests; the differences among multi-group data were tested with two-way ANOVA (treatment × genotype), followed by Bonferroni-Dunn post hoc tests. Planned within genotype comparisons within these data were analyzed by Student’s t tests. For each fly in the optical imaging experiments, the maximum fluorescent intensity change ratio before and after stimulation were calculated first. Then the ratio of vehicle-injected flies and ethanol-injected flies was analyzed by two-tailed, unpaired Student’s t tests. For t test and ANOVA, α = 0.05.The differences between two groups were tested with two-tailed, unpaired Student’s 582 residue in the C1 DAG binding domain of Munc13-1  = 10.321, p < 0.001; genotype, t = 4.058, p = 0.001). Ethanol concentrations of 25 and 50 mM significantly reduced FRET between the wild-type Munc13-1 C1 domain and dansyl-DAG . However, FRET between the Munc13-1E582A and dansyl DAG was not significantly altered by 25 and 50 mM ethanol . This resistance to ethanol meant that the mutant E582A C1 domain was significantly less sensitive to the effects of ethanol at both 25 and 50 mM concentrations as compared to the wild-type C1 domain  using optical physiology techniques. The OR42b neurons are excitatory cholinergic neurons that are strongly activated by ethyl acetate  = 21.474, p < 0.0001). As previously shown, injection of ethanol decreased the vesicle fusion in the OR42b OSNs elicited by ethyl acetate, however, the P84200Dunc13/+ flies were less sensitive to this presynaptic inhibition as compared to wild-type controls  = 9.19, genotype difference t = 0.583, p = 0.563; F = 11.16, genotype difference t = 0.037, p = 0.971; KK101383Dunc13 RNAi transgene throughout the nervous system using the n-Syb-Gal4 driver, also dramatically reduced the ethanol sedation sensitivity  = 72.73, p < 0.0001; 1/2 LOR for the experimental genotype, with Dunc13 RNAi being driven throughout the nervous system, was significantly longer than both the Gal4 driver control  and the Dunc13 RNAi control genotypes . The flies were viable, which suggested a partial knockdown of Dunc13 in neural tissue. Lastly, we induced the expression of the JF02440Dunc13 RNAi transgene postdevelopmentally in neurons with the elav-Gal4 and Gal80ts20 transgenes  = 5.191, p < 0.005). A 24-h 30°C induction of Dunc13 RNAi expression also leads to a significant decrease in ethanol sedation sensitivity compared to the within genotype control  = 9.875, p < 0.0001). The induced expression of this Munc13-1::EGFP transgene (48 h at 30°C) reduced the ethanol sedation sensitivity phenotype of P84200Dunc13/+ females . One possible reason for the lack of full complementation could be due to problems in trafficking the relatively large Munc13-1::EFGP protein to the presynaptic compartment. To see whether the Munc13-1::GFP protein was localized to the presynaptic compartment in Drosophila, we examined the neuromuscular junction of 3rd instar larvae with the genotype: UAS-Munc13-1::GFP/+; nsyb-Gal4/+. Munc13-1::EGFP was located within the presynaptic compartment in puncta. Bruchpilot, a protein localized to presynaptic active zones  can completely block the formation of rapid functional tolerance (tsshi alleles block synaptic vesicle recycling resulting in a depletion of the vesicle pool and a loss of presynaptic activity as well as other endocytosis-dependent events, such as receptor internalization (shi in the induction of tolerance (tsparalytic conditional allele in a voltage-gated sodium channel or temperature-sensitive alleles of the comatose NSF protein did not block the formation of functional tolerance. These data suggest that dynamics in the presynaptic vesicle pool during ethanol sedation, and not changes in presynaptic activity per se, are required for tolerance formation (There has been increasing evidence from olerance . At nonplization . When tholerance . Interesormation .cacophony voltage-gated Ca2+ channel and the cdk5 kinase, which are known to have important roles in presynaptic homeostatic responses to reduced synaptic activity (slowpoke increase the ability for hire frequency firing (+2 to increase the rate at which the RRP is replenished (Alcohol exposures that induce functional tolerance also bring about long-lasting changes in the levels of several presynaptic proteins that are required for the induction of functional tolerance . Some ofactivity . Mutantsactivity . Moreoveactivity . Increasy firing . This inlenished .582A, that inhibit ethanol binding to the C1 domain will reduce tolerance formation (In sum, there is strong evidence for specific homeostatic changes in presynaptic activity underlying ethanol tolerance, and an initial trigger for these changes may be ethanol inhibiting the binding of DAG to the C1 domain of Dunc13. A testable prediction from this hypothesis for the inhibition of Dunc13 as an initial event in the formation of tolerance is that compounds or mutations, such as the Munc13-1 Eormation . However


Many studies have used position-specific scoring matrices (PSSM) profiles to characterize residues in protein structures and to predict a broad range of protein features. Moreover, PSSM profiles of Protein Data Bank (PDB) entries have been recalculated in many works for different purposes. Although the computational cost of calculating a single PSSM profile is affordable, many statistical studies or machine learning-based methods used thousands of profiles to achieve their goals, thereby leading to a substantial increase of the computational cost. In this work we present a new database compiling PSSM profiles for the proteins of the PDB. Currently, the database contains 333,532 protein chain profiles involving 123,135 different PDB entries. Position-specific scoring matrices (PSSMs) have been used in many works to compute and predict a broad range of protein features. For example, PSSM profiles have been used to predict residue solvent accessibility , proteinSeveral resources compiling PSSM profiles are currently available. The Conserved Domain Database (CDD)  annotatehttp://3dcons.cnb.csic.es.In this work we present 3DCONS-DB, a database of PSSM profiles computed over protein sequences collected from the Protein Data Bank (PDB) . The maiCurrently, 3DCONS-DB database compiles PSSM profiles for 123,135 PDB entries, involving 333,532 protein chains and 83,297 non-redundant protein sequences. The database is freely available and accessible either through a browser or using a web service designed for programmatic access. In this section we also present the analysis and comparison of PSSM profiles in protein domain and non-domain regions and show how 3DCONS-DB data can be used to predict secondary structure and residue contact numbers.One of the aims of this work is to show that non-domains regions of proteins are functionally relevant and, therefore, having profiles characterizing non-domain residues (in addition to profiles of domain residues) will benefit the calculation and prediction of biological features in these regions. To that end, we have measured the occurrence of different biological features in protein domain and non-domain regions. Protein domains were determined in terms of the Pfam classification  for eachAnother important comparison is the quality and amount of information of PSSM profiles in domain and non-domain residues. To estimate these values, we analyzed the multiple sequence alignments (MSAs) that can be obtained by stacking the aligned sequences computed by PSIBLAST. For each PDB chain, PSIBLAST recovered an average number of 243 protein sequences that were used to generate a MSA. Pfam domains are computed from MSAs collected from non-redundant sets of protein sequences and, thus, the number of gaps is expected to be smaller in domain than non-domain positions. In our analysis we observed that the gap frequency was 1.8% in domain and 10.5% in non-domain positions, in agreement with the expected results. However, the gap frequency was 10 times greater in non-domain regions, we obtained an average number of 217 protein sequences that were aligned in these positions with no gaps. Then, to measure whether these aligned sequences contained more information than random alignments we computed the Williamson entropy  see Sec for the As a concrete example of how 3DCONS-DB has a clear impact in the agile development of new bioinformatics tools, we used 3DCONS-DB data to train a neural network classifier for protein secondary structure prediction. The selected neural network architecture was the same as the one described in PSIPRED , consistAs a second example of how 3DCONS-DB can simplify and facilitate the development of algorithms for the prediction of protein structural features, we have trained a support vector regression (SVR) model for predicting residue contact number (CN) using the same procedure as described in Yuan et al. . In theiThe evaluation consisted in a threefold cross-validation using different distance thresholds to define contacts between C-beta atoms. 3DCONS-DB is a new database that compiles PSSM profiles for PDB protein sequences with the aim set at facilitating the development and testing of prediction methods that use PSSMs. Currently, the database contains 123,135 PDB entries, involving 333,532 protein chains and 83,297 non-redundant protein sequences. The main difference with similar resources is that 3DCONS-DB annotates residues over whole protein sequences and not only on domain regions. However, the comparison of different biological features on domain and non-domain residues indicates that both types of protein regions seem functionally relevant. Indeed, non-domain residues are most often affected by post-translational modifications that domain ones and short linear motifs can be found more frequently in them as well, indicating that non-domain regions might be more involved in protein signaling than domain regions.Several biological features to characterize and compare protein domain and non-domain regions were collected from different sources. Secondary structure was calculated using the dssp software  for all k = 20) in order to ensure that the computed entropy value was not magnified due the class amino acid reduction.To measure the amount of information encoded behind PSSM profiles, we have calculated the Williamson entropy  values ohttps://www.sqlite.org) and a web server was built to dispatch the data. The web server was developed using the Ruby on Rails framework (http://rubyonrails.org) and was designed to collect and deliver PSSM profiles of PDB entries. 3DCONS-DB data can be accessed in three different ways: through a REST web service to retrieve PSSM profiles in JSON format, submitting a list of desired PDB ids and retrieving their PSSM scores in a ZIP file and, finally, using 3DCONS-DB web application to explore specific PSSM profiles through a browser.3DCONS-DB data was compiled computing the iterative BLAST algorithm (PSIBLAST)  with defhttp://d3js.org) and it summarizes PSSM scores for the entire selected sequence. Finally, the residue level PSSM table contains the exhaustive PSSM score list for each residue of the selected protein. 3DCONS-DB client can display the different levels of PSIBLAST information; thus, the scores of the different iterations, swapping between PSSM scores and position-specific frequency matrix scores, or exploring the PSSM scores for the different chains of a PDB entry.The web client was designed to display 3DCONS-DB data on a browser and to provide an interactive environment to explore PSSM profiles at sequential and structural level. The information is divided in three major panels see : the str


Background: The application of superparamagnetic particles as biomolecular transporters in microfluidic systems for lab-on-a-chip applications crucially depends on the ability to control their motion. One approach for magnetic-particle motion control is the superposition of static magnetic stray field landscapes (MFLs) with dynamically varying external fields. These MFLs may emerge from magnetic domains engineered both in shape and in their local anisotropies. Motion control of smaller beads does necessarily need smaller magnetic patterns, i.e., MFLs varying on smaller lateral scales. The achievable size limit of engineered magnetic domains depends on the magnetic patterning method and on the magnetic anisotropies of the material system. Smallest patterns are expected to be in the range of the domain wall width of the particular material system. To explore these limits a patterning technology is needed with a spatial resolution significantly smaller than the domain wall width.Results: We demonstrate the application of a helium ion microscope with a beam diameter of 8 nm as a mask-less method for local domain patterning of magnetic thin-film systems. For a prototypical in-plane exchange-bias system the domain wall width has been investigated as a function of the angle between unidirectional anisotropy and domain wall. By shrinking the domain size of periodic domain stripes, we analyzed the influence of domain wall overlap on the domain stability. Finally, by changing the geometry of artificial two-dimensional domains, the influence of domain wall overlap and domain wall geometry on the ultimate domain size in the chosen system was analyzed.Conclusion: The application of a helium ion microscope for magnetic patterning has been shown. It allowed for exploring the fundamental limits of domain engineering in an in-plane exchange-bias thin film as a prototypical system. For two-dimensional domains the limit depends on the domain geometry. The relative orientation between domain wall and anisotropy axes is a crucial parameter and therefore influences the achievable minimum domain size dramatically. Engineered magnetic domains with deliberately set magnetic properties and designed shapes in thin-film systems have proven to be useful in memory –2 and seThe size limit of patterning magnetic domains in continuous in-plane layer systems is expected to be in the range of the domain wall (DW) width, varying with material-specific magnetic parameters, but could not be tested yet. For exchange-bias material systems with in-plane anisotropy, typical DW widths are of the order of several hundreds of nanometers  to some 17Mn83 (30 nm)/Co70Fe30 (10 nm), as a prototype with unidirectional in-plane anisotropy , but it can be easily extended to other magnetic material systems.Thus, at present there is no method available where the lateral resolution is considerably higher as the expected minimum pattern sizes. Here we suggest mask-less patterning by the highly focused beam of a helium ion microscope (HIM), to lower the limits of ion beam induced magnetic pattering in continuous layer systems . The metMore specifically, the size limit of thermally stable engineered magnetic domains has been studied for prototypical domain geometries, and for varying magnetization directions with respect to the DWs. The prototypical EB system, with respect to saturation magnetization, magneto crystalline anisotropy and theoretically predicted domain wall width, was chosen to be fully accessed by a variety of quantitative characterization methods. The analysis of the patterns has been achieved by complementary experimental methods, characterizing the magnetization profile by X-ray photo emission electron microscopy (X-PEEM) and investigating the magnetic charge state of the DWs by magnetic force microscopy (MFM). The experiments have been corroborated by micromagnetic simulations.The ion bombardment induced magnetic pattering of artificial domains in exchange-bias multilayers is fundamentally based on two energy-transfer mechanisms from the ions to the material system. The predominant effect, the electronic energy transfer , causes a reorientation of the local unidirectional anisotropy. The second and considerably weaker effect is the nuclear energy transfer causing defects in the atomic lattice structure . These dThe area modified by an ion beam is defined by the beam diameter and the ion straggling in the sample. The corresponding lateral range of this effect has been estimated by SRIM  simulati between fixed antiparallel unidirectional anisotropies of adjacent domains and the DW normal vector   and tail-to-tail (tt) domain configurations ( = 0°) to bipolar for the side-by-side (ss) configuration ( = 90°). The DW charge state in the xy-plane, parallel to the F layer, was characterized by MFM  measurements. Two different sensitivity directions have been chosen:  obvious ,g. The a obvious . = 90°,  = 30° , it is evident that for b ≤ 2 μm there is a significant crosstalk between neighboring DWs. However, the smallest distinguishable periodic magnetic patterns are observable in b = 500 nm. In earlier experiments using masks, a critical domain width of 700 nm has been found for non-periodic domain patterns with hh and tt in a Fe50Mn50 (10 nm)/Ni81Fe19 (5 nm) layer system [81Fe19 is smaller than that of Co70Fe30, it is the small saturation magnetization Ms together with a thinner F layer that potentially allows for the observation of smaller DW tails in this material system  and therefore smaller thermally stable domains. The limitations correlated to the patterning process when using shadow masks, however, cause spatial broadening of the ion dose gradient. Since the concerned regions correspond to the DW regions, there is a strong impact on the actual DW fine structure due to both geometrical and magnetic deviations from the set structure leading to a larger minimum domain size.The magnetic charge contrast of this pattern obtained by MFM is shown in b ≤ 2 μm . Since tr system . Althougd of 10 μm, 7.5 μm, 5 μm, 2.5 μm, 2 μm, 1 μm and 500 nm, comprise the previously investigated different angles  and different DW–DW distances. The domain patterns were analyzed by MFM and simulated using the object-oriented micromagnetic framework (OOMMF) [To determine the ultimate size limit of the prototypical EB-system, the results of the previous studies were merged by a series of experiments with two-dimensional domains of three fundamental shapes. These shapes, namely squares, circles, and equilateral triangles, with deliberately set edge lengths or diameters  (OOMMF)   to  = 90° (upper and lower border) and back. For the equilateral triangles two exemplary patterns have been realized: one with the symmetry axis parallel  also appears distorted in the MFM data and the different DW types appear blurred. In the simulations, such structures are clearly visible and blurring of DWs appears at d = 1 μm. Again, the reason for the discrepancy between measurement and simulation can be attributed to local fluctuations of material parameters resulting from the polycrystallinity of the layer system and the sample–tip distance. Also, the magnetization profile of the MFM tip was neglected in the simulations.For charged DWs it is a priori not possible to correlate the MFM signal to the magnetization configuration, as there is no one-to-one correspondence of these two quantities . Therefo; instead the domain shows a curled magnetization state. Magnetic charges of inverted polarity are, therefore, close together and cannot be detected by the MFM tip, averaging over a certain lateral range defined by the tip radius and the distance to the sample surface.Since simulations and measurements are in very good qualitative agreement concerning DW types and distortion, the simulated magnetization distributions –o are usThe DW profile obtained for circular domains in the simulations also qualitatively reproduces the measurement. The simulations underestimate the smallest stable domain size because of the discussed reasons. In contrast to the triangular domains, the minimum size of circular domains is smaller: as the DW charge state continuously changes along the domain boundary corresponding to the radial rotation of the DW normal vector, the resulting demagnetization field changes as well. Thus, the torque that results from the interaction between the demagnetization field and the magnetization distribution of the domain decreases radially and leads to a more uniform orientation of the magnetization field ,n withind = 2 μm, with already strong alteration of the set domain shape. This value is, for the abovementioned reasons, smaller than experimentally found (d = 5 μm).For the triangular domains ,d, the o ≠ 0° or  ≠ 90°. The smallest domain size was found for square and circular structures to be 2 µm.By employing the helium ion beam of a scanning helium ion microscope defocused to 8 nm for mask-less ion bombardment induced magnetic patterning a prototypical in-plane exchange-biased layer system has been modified locally with a resulting patterned spot of less than 40 nm diameter at the AF/F interface. The narrow beam diameter enabled lateral magnetic modifications of the continuous layer system well below currently available light ion patterning techniques and well below expected stable domain sizes. It was shown that the domain wall width is strongly connected to the angle between unidirectional anisotropy and domain wall normal. Additionally, the influence of the domain wall overlap on the domain stability was quantified. For magnetic-domain engineering, this method enables strategies to fabricate domains of minimum size. It was shown that the minimum domain size for magnetic stripes with head-to-head magnetization configuration in the chosen prototypical system is at least 500 nm. For two-dimensional domains, the minimum stable domain size depends on the domain shape and the interplay of both domain and domain wall charges, corresponding stray fields and local anisotropies. For the presently investigated thin film system with in-plane anisotropy, key enablers to achieve minimum domain sizes are rounded vertices to support continuous charge transitions, the avoidance of monopolar charges and of DWs with This method allows for the magnetic patterning via kiloelectronvolt light ion beams of a variety of material systems in order to test for fundamental properties governing minimum achievable domain sizes.17Mn83 (30 nm)/Co70Fe30 (10 nm) was grown by RF sputtering at a power of 160 W and an argon gas flux of 155 sccm on a naturally oxidized 5 nm × 5 nm Si(100) wafer, with Cu (5 nm) buffer and Ta (10 nm) capping. EB at the interface between the antiferromagnetic (AF) and ferromagnetic (F) layer has been initialized by heating at 573 K for 90 min and subsequent cooling at a rate of 1 K·min−1 for 300 min to room temperature in an external magnetic field of 80 kA·m−1.The prototypical in-plane EB layer system Ir−1 during ion bombardment. The samples were aligned with their initial EB-field direction pointing antiparallel to the external magnetic field of the holder. 15 keV helium ion bombardment was performed on an area of 500 μm × 500 μm consisting of 216 × 216 separate points. To do so, the ion beam was defocused, leading to a probe diameter of 8 nm. The resolution was determined by the knife-edge method from the image sharpness [15 ions·cm−2 to induce a maximum change of HEB [A commercial HIM (Zeiss Orion Plus) has been modified with a sample holder allowing for the application of an in-plane magnetic field of 95 kA·mharpness . The ione of HEB . A Raith−1 were employed.MFM measurements were performed by a Nanosurf Flex-AFM with C3000 controller in tapping/lift mode with a lift height of 80 nm, a peak-to-peak amplitude of 80 nm and a pixel size of 20 nm. These settings were chosen from preliminary experiments as a trade-off between lateral charge-contrast resolution and minimal signal overlap from the sample topography. Additionally, an SIS ULTRAObjective in non-contact/lift mode with a lift height of 100 nm and a pixel size of 200 nm was applied for the MFM measurements. Hard magnetic MFM probes (Nanosensors PPP-MFMR) with a nominal resonance frequency of 70 kHz and a spring constant of 2.8 N·m in the F layer of the EB layer system were performed at beamline UE56/1-SGM of the synchrotron radiation facility BESSY II after excitation with right and left circularly polarized X-rays with an energy of 709 eV (Fe L3 edge). Prior to measurements, the capping layer was thinned by argon ion sputtering allowing the Fe photoelectrons to escape from the layer system. The XPEEM measurements were carried out in grazing incidence of the incoming synchrotron radiation while the angle between the photon -vector surface projection  and the initially set EB field direction of the thin film was varied. Measurements of partial electron yield maps were imaged with a 43 μm field of view. One image pixel represents a sample area of 65 nm × 65 nm. Of particular note is that the XMCD signal Δ of the partial electron yield of the two respective helicities (Δ = (Iσ+ − Iσ−)/(Iσ+ + Iσ−)) is proportional to .XPEEM measurements –49 of th70Fe30 film assuming a uniaxial magnetic anisotropy constant of KF,NB = 4.5 × 104 J·m−3 for the non-bombarded areas [KF,B = 0.71KF,NB for the bombarded areas [Ms,NB = 1226 kA·m−1 and Ms,B = 1175 kA·m−1 for the ion-bombarded regions [A = 3 × 10−11 J·m−1 [HEB,NB = 12.3 kA·m−1) and bombarded areas  has been mimicked by a local magnetic field the field direction of which corresponds to the direction of the unidirectional anisotropy of the domain. EB fields were determined by Kerr microscopy of the engineered domain pattern. The mesh size of 10 nm × 10 nm was chosen to follow the stray-field exchange length of the film of 6 nm [ between the unit magnetization direction  of one mesh element and the local total magnetic field  starting from an ideal alignment of the set magnetization direction along the EB field direction. Note that on basis of experimental findings Ms was chosen differently for bombarded and non-bombarded areas [ or a maximum step number of 2 × 105 iterations.Micromagnetic simulations were carried out using the object-oriented micromagnetic framework (OOMMF) for a 10 nm thick Coed areas  and KF,Bed areas . The sat regions . The exc11 J·m−1 . The EB  of 6 nm ,51. Baseed areas . The stoSRIM simulations of the ion energy loss distribution: To determine the distribution of ions and the spatial distribution of the transferred energy in the sample, simulations using the SRIM software framework have been performed [Si = 2.32 g·cm−3 [ = 2.65 g·cm−3 [Cu = 8.92 g·cm−3 [ = 8.386 g·cm−3 [ = 8.565 g·cm−3 [Ta = 16.65 g·cm−3 [ of binary alloys with AnB1−n stoichiometry were approximated from the density values of pure metals usingerformed . The lay2 g·cm−3 ,  = 2.655 g·cm−3 , ρCu = 82 g·cm−3 ,  = 8.386 g·cm−3 ,  = 8.565 g·cm−3 , ρTa = 15 g·cm−3 . The denx = n/100. The SRIM compound correction was deactivated for all layers except SiO2.with x being the mole fraction, 6 helium ions with a kinetic energy of 15 keV entering the layer system orthogonally to the surface. The resulting penetration depth z and the lateral x/z distributions were extracted from the IONZ3D file of the program (z-axis Θz(x) was approximated by a Gaussian normal distribution having the form:Simulations were performed using the monolayer collision mode for 2 × 10 program . The latAz represents the energy loss per depth unit, σz is the standard deviation of the normal distribution.db(z) in the material system is approximated by the 2σ value of the respective normal distribution, db(z) = 4σz.Since the reorientation of the unidirectional EB anisotropy is attributed to local hyperthermal heating by the electronic interaction of the ion beam with the layer system , the effAz(z). The effective beam diameter and the energy loss at the common F/AF-interface where the exchange bias is located are db(20 nm) = 27 nm and Az(20 nm) = 109 eV·nm−1·ion−1, and at the AF/buffer-interface db(50 nm) = 92 nm and Az(50 nm) = 81 eV·nm−1·ion−1.The simulations show a continuous broadening of the ion beam in the material system and simultaneously a decrease of the transferred energy per depth unit Definition of the angle: describes the angle between the unidirectional anisotropy axis  of the non-bombarded domain and the domain wall normal   was modand0 = 4π × 10−7 N·A−2 is the vacuum permeability, and tf is the thickness of the FM layer. The saturation magnetization Ms as a function of the ion dose was measured in [Ms,NB = 1226 kA·m−1, Ms,B = 1175 kA·m−1 [KF a a function of the ion dose was extracted from a fit function in [KF,NB = 4.5 × 104 J·m−3 [KF,B = 0.71KF,NB [HEB,NB = 12.3 kA·m−1 and HEB,B = 10.0 kA·m−1 were determined from Kerr-microscope measurements. With these values the domain wall tail lengths Dtail,NB = 1.04 μm and Dtail,B = 1.32 μm were calculated for the prototypical Co70Fe30/Ir17Mn83 layer system. For the Ni81Fe19 layer system from [KF,NB = KF,B = 2.3 × 102 J·m−3 [Ms,NB = Ms,B = 780 kA·m−1 [HEB,NB = −15.5 kA·m−1 and HEB,B = 13.9 kA·m−1 [Dtail,B = 972 nm in the bombarded and Dtail,NB = 874 nm in the non-bombarded regions.to describe the domain wall tail length of the bombarded and non-bombarded regions individually. Here, γ ≈ 0.577 is the Euler constant, μsured in  for a si5 kA·m−1 ). The F ction in . The rel04 J·m−3  and KF,B.71KF,NB . The exctem from , the val02 J·m−3 , Ms,NB =0 kA·m−1 , HEB,NB 9 kA·m−1  were use


Tetratricopeptide repeat (TPR) proteins belong to the class of α-solenoid proteins, in which repetitive units of α-helical hairpin motifs stack to form superhelical, often highly flexible structures. TPR domains occur in a wide variety of proteins, and perform key functional roles including protein folding, protein trafficking, cell cycle control and post-translational modification. Here, we look at the TPR domain of the enzyme O-linked GlcNAc-transferase (OGT), which catalyses O–GlcNAcylation of a broad range of substrate proteins. A number of single-point mutations in the TPR domain of human OGT have been associated with the disease Intellectual Disability (ID). By extended steered and equilibrium atomistic simulations, we show that the OGT-TPR domain acts as an elastic nanospring, and that each of the ID-related local mutations substantially affect the global dynamics of the TPR domain. Since the nanospring character of the OGT-TPR domain is key to its function in binding and releasing OGT substrates, these changes of its biomechanics likely lead to defective substrate interaction. We find that neutral mutations in the human population, selected by analysis of the gnomAD database, do not incur these changes. Our findings may not only help to explain the ID phenotype of the mutants, but also aid the design of TPR proteins with tailored biomechanical properties. Due to their extended water-exposed surface and high degree of flexibility, they play a particularly important role in the formation of multiple protein–protein binding interactions. α-solenoid domains consist of arrays of repetitive α-helical units with variations in the numbers of repeats and the precise spatial arrangement of the helices . GloballIn the case of HEAT repeat domains, simulations have previously shown that the conserved hydrophobic core formed by part of this consensus sequence confers fully reversible, spring-like elasticity to the domains . Armadil4-L7-G8-Y11-A20-F24-A27-P32 , which includes three additional positions compared to most other TPR repeats  and two partially resolved repeats. We used a moving harmonic potential of ~1.25 kcal mol−1 Å−2, attached to the C-terminal end of the TPR domain (TPR11), at a velocity of 1 Å ns−1 to increase its separation from the fixed N-terminus (TPR2) and thereby elongate the domain. We then extracted four independent extended conformations obtained from the trajectory under force and allowed the OGT-TPR domain to relax its conformation in further unbiased simulations.To characterise the elasticity of the OGT-TPR domain and to ascertain if its folded structure remains intact upon enforced elongation, we performed steered molecular dynamics (SMD) simulations on wild-type (wt) OGT-TPR  and the As shown in The results thus show that the OGT-TPR superhelix displays spring-like mechanical behaviour. Enforced extensions or distortions of the structure lead to a loading of this protein nanospring, by which energy is stored in the elongated conformation without disrupting its secondary structure or intramolecular contacts. Upon release of the driving force, the elongated superhelix elastically relaxes to its original ground state, thereby releasing the energy that was previously stored.SMD simulations of the ID-related mutants  Fig. 2B.3To accurately determine the spring constant of the domain and thereby obtain the energy required for its elastic deformation, we conducted further equilibrium MD simulations. The wt and mutant OGT-TPR domains were each simulated for a total time of 2 µs, combining data from four replicates of 500 ns length. spring = kBT / σ2k, the width σ of the normal distribution (its standard deviation) gives rise to a spring constant of WTk = 20.80 ± 0.07 pN/nm  while the shorter population shows a markedly softened spring constant of L254F2k = 12.61 ± 0.08 pN/nm. For the R284P mutant, the lengthened population also reflects a softer nanospring with a spring constant of R284P2k = 17.02 ± 0.13 pN/nm, while the major population remains close to the wild-type (R284P1k = 19.92 ± 0.01 pN/nm). By contrast, the narrower distribution of lengths observed for the A319T variant emerges due to a rigidified nanospring (A319Tk = 24.90 ± 0.11 pN/nm).The major species of the L254F mutant has a spring constant similar to the wt , as previously reported in Gundogdu et al.  at position X1 ± 1.0° G, 4D. Th1 ± 1.0° B. In theof R284P C. The su5To differentiate mutations that lead to pathological phenotypes from neutral mutations that are observed in humans but are not related to disease, we selected two further OGT-TPR variants (I279V and A310T), which are unlikely to lead to aberrant OGT function. In addition, we probed an alternative OGT-TPR variant at position 254 (L254I) to investigate if other mutations at this site give rise to a distortion of the TPR nanospring similar to that observed for L254F . L254F rCandidates for control mutagenesis unlikely to cause disease phenotypes were selected from OGT variants present in the Genome Aggregation Database (gnomAD) . The gnoI279V k= 21.40 ± 0.07 pN/nm (I279V) and A310Tk = 20.31 ± 0.09 pN/nm (A310T). These results show that the neutral mutations neither alter the global conformation nor the biomechanical behaviour of the OGT-TPR domain.As seen in L254Ik = 22.99 ± 0.08 pN/nm  was chosen to model the wt and mutant OGT-TPR domains. This model protein includes repeats TPR2 to TPR11. The constructs were capped and solvated in a triclinic box of 116.6 × 116.6 × 116.6 Å. Na+ and Cl- ions were added to neutralise the system and reach a physiological concentration of 0.15 M NaCl. The amber99sb-ildn force field , using an integration time-step of 4 fs.on 5.1.5 . For eacThe temperature was kept constant by weakly coupling (t = 0.1 ps) the protein and solvent separately to a temperature bath of 298 K with the velocity-rescale thermostat of Bussi et al. . The preAnalysis of the trajectories and estimation of the spring force constant. We used MDAnalysis  using a force constant of 10 kJmol-1Å−2 and applying a pulling potential of 5 kJmol-1Å−2 to displace helix TPR12A  at a constant velocity of 1 Å ns−1 in the z-direction.ulations . To ensuThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Roseobacter clade. Superoxide levels outside the cell are controlled via a tightly regulated balance of production and decay processes in response to cell density and life stage in batch culture. Removal of extracellular superoxide leads to substantial growth inhibition. These findings point to an essential role for superoxide in the health and growth of this ubiquitous group of microbes, and likely beyond.Formation of reactive oxygen species (ROS) through partial reduction of molecular oxygen is widely associated with stress within microbial and marine systems. Nevertheless, widespread observations of the production of the ROS superoxide by healthy and actively growing marine bacteria and phytoplankton call into question the role of superoxide in the health and physiology of marine microbes. Here, we show that superoxide is produced by several marine bacteria within the widespread and abundant  Roseobacter clade and specifically target the link between extracellular superoxide and physiology for two species. We reveal for all species a strong inverse relationship between cell-normalized superoxide production rates and cell number. For exponentially growing cells of Ruegeria pomeroyi DSS-3 and Roseobacter sp. strain AzwK-3b, we show that superoxide levels are regulated in response to cell density through rapid modulation of gross production and not decay. Over a life cycle of batch cultures, extracellular superoxide levels are tightly regulated through a balance of both production and decay processes allowing for nearly constant levels of superoxide during active growth and minimal levels upon entering stationary phase. Further, removal of superoxide through the addition of exogenous superoxide dismutase during growth leads to significant growth inhibition. Overall, these results point to tight regulation of extracellular superoxide in representative members of the Roseobacter clade, consistent with a role for superoxide in growth regulation as widely acknowledged in fungal, animal, and plant physiology.There is a growing appreciation within animal and plant physiology that the reactive oxygen species (ROS) superoxide is not only detrimental but also essential for life. Yet, despite widespread production of extracellular superoxide by healthy bacteria and phytoplankton, this molecule remains associated with stress and death. Here, we quantify extracellular superoxide production by seven ecologically diverse bacteria within the  All aerobic organisms form ROS intracellularly as a by-product of respiration or photosynthesis. ROS, particularly superoxide and hydrogen peroxide, are also produced extracellularly by a broad range of cells, including animals, fungi, phytoplankton, and bacteria (–Reactive oxygen species (ROS) are short-lived oxygen radicals that form as intermediates in the reduction of oxygen to water. The ROS superoxide  and assembled into a tree using the interactive Tree of Life  (http://itol.embl.de/). Sequence similarity is only an estimate of evolutionary relationships. Download FIG S1, PDF file, 0.1 MB.Neighbor-joining phylogenetic tree based on 16S ribosomal DNA sequences of seven Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02668-18.2FIG S2FIG S2, PDF file, 0.2 MB.Simplified schematic of the flow injection chemiluminescence approach used to measure extracellular superoxide in this research. Download Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02668-18.3FIG S3Roseobacter clade species. Here we illustrate the results of one loading experiment as an example of the range of trends observed. Download FIG S3, PDF file, 0.1 MB.Typical steady-state superoxide concentration trends as a function of cell load in mid-exponential cultures of seven Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02668-18.4TABLE S1Roseobacter clade bacteria investigated in this study. Download Table S1, PDF file, 0.1 MB.Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02668-18.5TABLE S2Roseobacter species. Download Table S2, PDF file, 0.1 MB.Compilation of superoxide measurements for cell loading experiments with seven Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Roseobacter clade cultures. Briefly, mid-exponential-phase cells of Ruegeria pomeroyi DSS-3 and Roseobacter sp. strain AzwK-3b were diluted 10- and 100-fold into sterile seawater and allowed to acclimate for 6 h, and then extracellular superoxide production was analyzed by loading a single aliquot (one of two volumes—0.1 or 1.0 ml) from each dilution onto in-line filters. This approach circumvents potential stress imposed on the cells during the sequential loading processes, which involved the addition of an increasing number of cells onto the filter, combined with intermittent flow. Consistent with results from the sequential loading method  responsible for extracellular superoxide production within marine bacteria is currently limited to Roseobacter sp. AzwK-3b, which utilizes an animal heme peroxidase (AHP) that is found within the outer membrane and cell-free exudate (2O2-degrading) and oxidase (O2•−-producing) activity that is controlled at times by extracellular hydrogen peroxide levels . In R. pomeroyi DSS-3, these results could not be accounted for by the addition of heat-killed SOD, indicating that the growth inhibition response was specific to the active enzyme, and thus, the degradation of extracellular superoxide  at 20°C on an orbital shaker (150 rpm)  (Santa Cruz Biotechnology) (2) standard to aged filtered natural seawater influent, measuring the chemiluminescent decay over time, and modeling the decay data using a pseudo-first-order decay function (2 standard curve (Axenic cultures of bacteria within the 150 rpm) . Cells whnology)  as previhnology) , 18 and function . Furtherrd curve .Roseobacter clade  prepared in filter-sterilized (0.2 μm) 75% natural seawater (K-NSW) on an orbital shaker (150 rpm) (Ruegeria pomeroyi DSS-3 were collected from replicate (n = 3) flasks along a growth curve to measure superoxide through a life cycle. Optical density at 600 nm (OD600) values were converted to cell numbers via flow cytometry analysis (see below). At each sampling point, an aliquot of cells was fixed with a final concentration of 0.5% glutaraldehyde and stored at 4°C in the dark for later flow cytometry analysis.Seven bacterial species within the 150 rpm) . TriplicR. pomeroyi DSS-3 and Roseobacter AzwK-3b were subsampled and diluted 10× and 100× in filter-sterilized natural seawater in 250-ml flasks. Undiluted subsamples were also transferred to new sterile 250-ml flasks in equal volumes as diluted cultures to maintain consistency between cultures. The transfers were shaken at 150 rpm on an orbital shaker for 6 h and then sampled for superoxide production. Cells after incubation were fixed with a final concentration of 0.5% glutaraldehyde and stored at 4°C in the dark for later cell enumeration via flow cytometry.Mid-exponential-phase cultures of A subset of incubations contained additions of superoxide dismutase (bovine)  added from a 10-kU/ml stock solution. SOD was inactivated by heating to boiling and then incubating at 100°C for 30 min. The activity of the boiled SOD was assessed by running a superoxide calibration (see below), followed by the addition of the inactivated SOD to confirm that the signal was not impacted. Active SOD was then added to confirm the superoxide signal.−1) for 3 min. Bacterial cells were counted based on diagnostic forward scatter versus green fluorescence signals. Instrument-specific beads were used to calibrate the cytometer.Cell counts were conducted on a Guava EasyCyte HT flow cytometer (Millipore). Samples were diluted (1:100) with filtered seawater (0.01 µm). Samples and filtered seawater blanks were stained with SYBR Green I (Invitrogen) according to the manufacturer’s instructions and incubated in a 96-well plate in the dark at room temperature for at least 30 min. Samples were analyzed at a low flow rate  as described in detail previously . Extracellular superoxide was measured by running aged, 0.2-μm-filtered natural seawater past the filter-supported colonies directly into the instrument where it was mixed with the superoxide-specific chemiluminescent probe methyl −1) using a peristaltic pump until they converge in a spiral flow cell immediately adjacent to a photomultiplier tube, which continuously acquires data that is displayed in real time using a PC interface. Similar systems have been used to generate high-sensitivity measurements of natural superoxide concentrations and decay rates  were prepared in NaOH (pH 12.5) amended with 50 to 100 μM diethylene-triaminepentaacetic acid (DTPA) in order to sequester trace contaminants that would otherwise significantly reduce the lifetime of superoxide. Superoxide concentrations in primary standards were quantified by measuring the difference in absorbance at 240 nm before and after the addition of superoxide dismutase (SOD) (∼2 U ml−1) and then converting to molar units based on the molar absorptivity of superoxide corrected for the absorption of hydrogen peroxide formed during decay at the same wavelength . Baseline correction was achieved by subtracting the average background signal generated from the AFSW passing over the in-line filter, without KO2, and reacting with the MCLA reagent for at least 1 min, as described above. Baseline-corrected chemiluminescence data collected over several minutes of superoxide decay in standard solutions were log linear and therefore modeled using pseudo-first-order decay kinetics.To prepare calibration curves, the chemiluminescence signal generated from the secondary standards was baseline corrected for chemiluminescence signal arising from the autooxidation of the MCLA reagent and extrapolated back to the time at which the primary standard was diluted (R2 > 0.9), with sensitivities ranging from 0.6 to 3.1 chemiluminescence units per pM superoxide.Daily calibration curves were generated from three paired observations of time zero superoxide concentration (dependent variable) and extrapolated chemiluminescence (independent variable) using linear regression. Because chemiluminescence values were baseline corrected, regression lines were forced through the origin. Calibrations yielded highly linear curves . Production rates of superoxide by each population were normalized to the total number of cells added to provide cell-normalized rates  (n = 3) were conducted for superoxide measurements using equivalent cell densities.For biological experiments, as in calibration runs, a clean syringe filter  was placed downstream of the peristaltic pump and upstream of the flow cell in the analyte line, where it remained throughout the duration of the experiment. Stable baseline signals (<4% coefficient of variation) were generated in biological experiments from AFSW passing over the in-line filter and reacting with MCLA for at least 1 min. The pump was temporarily stopped, and cells were added to the in-line filter using a syringe to the desired cell density. Microscopic examination of the cells after analysis did not indicate visible cellular damage. The presence of cells did not alter flow rates during the experiment, as flow rates after cell addition were typically within the analytical error of the rate determined beforehand. Extracellular superoxide produced by the organisms housed on the in-line filter and released into the AFSW carrier solution was detected downstream upon mixing with the MCLA reagent in the flow cell. Chemiluminescence signals yielded in biological experiments were considered stable upon achieving a coefficient of variation (CV) equal to or less than that of the baseline for at least 1 min (CV ≤ ±4%). These signals were corrected for background chemiluminescence by subtracting the average baseline obtained immediately before the addition of cells (without SOD added) and converted to steady-state concentration measurements using the sensitivity measured in that day’s calibration (see above). The detection limit for these measurements, calculated assuming that the minimum detectable baseline-corrected signal was three times the standard deviation of the baseline, was 85 ± 19 pM. Net superoxide production rates were then calculated as the product of the steady-state superoxide concentration and flow rate   and S3. 10.1128/mBio.02668-18.6TABLE S3Ruegeria pomeroyi DSS-3 batch culture life cycle experiments. Download Table S3, PDF file, 0.1 MB.Superoxide data for Copyright © 2019 Hansel et al.2019Hansel et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the k in seconds−1) were determined by modeling the log-transformed decay data with pseudo-first-order kinetics.To calculate superoxide decay by bacteria, standard additions of superoxide were added to a subset of the cell cultures . After s−1) was added to the AFSW at the end of each run. SOD always caused a rapid decrease in signal to a final baseline that was typically below the initial baseline measured before cells were loaded. The difference in the initial and final baselines (∼200 chemiluminescence units) was of the same magnitude as the decrease in baseline observed when the same amount of SOD was added to the carrier solution in the absence of cells. This baseline decrease reflects either a small, yet nonzero concentration of superoxide in the AFSW carrier solutions and/or (more likely) an effect of SOD on the background chemiluminescence produced by the autooxidation of MCLA  was conducted with α = 0.05. The ANOVA was followed by a Tukey test of honestly significant differences (HSD) with


Mycobacterium avium subsp. paratuberculosis (MAP) ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands. Analyzed herds were representative for location and herd size of dairy goat herds in the Netherlands. A significantly higher proportion of the analyzed 49 herds were organic as compared with the total Dutch dairy goat population. First, the MAP ELISA was optimized using 992 paired serum and milk samples. At a cut-off of 25 S/P%, the relative sensitivity (Se) was 58.4%  and relative specificity (Sp) was 98.5% , as compared to serum ELISA results. The percentage of positively tested herds was 78.2% . The percentage of positive milk samples per herd (n = 22) was on average 4.6% . Average age of ELISA-positive (3.2 years) and -negative goats (3.2 years) was not different. Significantly more vaccinated goats tested positive (6.7%) as compared with nonvaccinated goats (1.1%). This study shows that a high number of vaccinated and nonvaccinated commercial dairy goat herds in the Netherlands have MAP-ELISA-positive goats.The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated  Mycobacterium avium subsp. paratuberculosis (MAP) [Paratuberculosis is a chronic enteritis of ruminants caused by is (MAP) . The disis (MAP) ,3,4.Several diagnostic methods are currently available to detect paratuberculosis, including agent detection by PCR or fecal culture and antibody detection by, for example, ELISA or agar gel immunodiffusion test (AGIDT). Comparing the accuracy of these different tests is difficult and depends on the goal for which the results are used ,6,7,8. T® . However, limited information is available on the effect of vaccination on ELISA test results in goats, as well as on the boostering effect due to subsequent infection. Therefore, it is difficult to distinguish antibody responses due to natural infection from responses due to vaccination.Vaccination is a widely used management measure to control paratuberculosis. Meta-analyses have shown a positive effect of vaccination on delaying or reducing production losses and pathogenic effects in the majority of studies ,15. A vaSeveral global prevalence studies in goats showed low animal prevalence (<16%) ,20,21,22The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated MAP ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands.www.skal.nl). In 2013, 49 dairy goat herd farmers submitted 9205 milk samples to GD Animal Health, of which most samples were collected to confirm suspected or clinically affected goats. At the request of GD Animal Health, 2464 out of these, originating from 22 conventional herds, were randomly sampled. Besides 992 milk samples, 20 farmers submitted a paired serum sample from paratuberculosis-suspected goats.The dairy goat industry in the Netherlands is increasing but was relatively small in 2013, with 400,000 goats kept in 333 herds with more than 50 goats . SamplesMycobacterium phlei extract (milk dilution 1:2 and serum dilution 1:20). In the indirect ELISA, specific MAP antibodies were bound by an anti-ruminant IgG conjugate. Results were expressed as S/P%, which was calculated as follows: / × 100. As no data were available for caprine milk samples, GD Animal Health calculated the most appropriate cut-off value using the abovementioned 992 paired caprine milk and serum samples. The relative sensitivity and specificity of the ELISA for caprine milk samples were calculated in comparison to the serum samples, in absence of a golden standard.All serum and milk samples were tested using an ELISA with a relative sensitivity (Se) and relative specificity (Sp) of 64.7% and 99.2%, respectively, for bovine, ovine, and caprine serum samples as compared to fecal culture . A specific evaluation of caprine serum samples showed a sensitivity of 82% in a group of infected goats, based on tissue culture or histopathology, and 55% in a group of negatively tested goats based on fecal culture, as well as an overall specificity of 96% . The ELIwww.cbs.nl, January, 2014). Representativeness for location and type  of the study herds was tested using a proportion test. Representativeness for herd size of the study herds was tested with a median test. For all herds for which the percentages of positive goats were estimated, farmers were asked to provide information about vaccination (yes/no and more/less than one year ago) and the age of the goats. Differences in average age and vaccination status between positively and negatively tested goats were tested using a t-test and a proportion test, respectively.Locations of herds at the province level in the Netherlands were available for all 333 Dutch dairy goat herds with more than 50 goats, herd sizes of 253 herds were known, and 60 out of these 333 herds were organic (Statistics Netherlands (CBS), Herds were classified as positive if at least 1% of the milk samples tested positive, regardless of their vaccination status. Sampling weights were considered to determine the percentage of positive herds and to adjust for the fact that the proportion of organic herds in the study population was larger than the proportion of organic herds among all dairy goat herds in the Netherlands. Sampling weights were calculated as the inverse of the herd’s sampling probability, which was calculated as the number of organic herds in the study population in relation to the number of organic herds in the sampling population. All statistical analyses were performed using STATA 13.1 software .The study described in this manuscript was conducted in compliance with legislation on animal use and practicing veterinary medicine in the Netherlands. This was an epidemiological study in the field using common sampling methods for routine diagnostic purposes. According to Dutch legislation, such studies do not need approval from an animal ethics committee, but they need to be performed according to the Dutch Veterinary Practice Act. n = 992, 95% CI: 48.8–67.6%) and the Sp was 98.5%   . The levp > 0.05). Average herd sizes  based on goats older than one year were 856  and 945 , respectively, and these were not significantly different from a median herd size of 668 goats older than one year in the 253 Dutch dairy herds with a known herd size . The percentage of organic dairy goat herds in the analyzed 49 herds (32.7%) was significantly higher than the percentage of organic dairy goat herds in the Netherlands . For that reason, sampling weights were used to determine the percentage of positive goat herds. Only one organic dairy goat herd submitted randomly collected samples, and therefore, the percentage of positive goats per herd was only estimated for 22 conventional dairy goat herds.The distribution of all participating herds per province was not significantly different from the average distribution of dairy goat herds in the Netherlands . There was no significant difference between the percentage of positively tested conventional and organic herds . In all 27 herds in which vaccinated goats were present, at least 1% of milk samples was positive. All negatively tested herds were not vaccinated.In total, 9205 milk samples were tested; mean number of samples  per herd was 188 . The percentage of positively tested herds corrected for the proportion of included organic herds was 78.2% .The percentage of MAP ELISA positively tested goats per herd, based on 2464 randomly collected milk samples from 22 conventional herds with on average 112 samples per herd, was on average 4.6%  . The pert-test, p > 0.05).The age of all animals from 18 herds was known. Two out of these 18 herds, with only negative test results, were excluded. The average  ages of the positively and negatively tested goats from the remaining 16 herds were 3.2  and 3.2 , respectively, which was not significantly different (p < 0.01). Of the goats that were vaccinated less than a year before, 6.3% tested positive, and of 1470 goats that were vaccinated more than a year before, 6.7% tested positive, which was not significantly different (For 2418 out of 2464 goats (93.8%), vaccination status was known; 1.1% of 900 nonvaccinated goats tested positive and 6.7% of 1518 vaccinated goats tested positive, which was significantly different  ; CroatiaIn the Netherlands, the most frequently used MAP test is an ELISA because of the relatively low costs and high throughput. A disadvantage is the relatively low sensitivity; a low and delayed antibody response hampers early detection of infected animals ,9,36. AnLimited data are available on the effect of vaccination on MAP ELISA results in goats, although goat kids vaccinated at 5 days or 5 months of age had a maximum antibody response at 30 days postvaccination . ELISA rThe ages of positively and negatively tested goats were not different. Another study showed that mean S/P% in milk is higher in late lactation than at kidding; however, test performance is comparable across lactational stages .In conclusion, the results of this study show a high percentage of vaccinated and nonvaccinated commercial dairy goat herds with at least one MAP-ELISA-positive goat in the Netherlands. Further research on ELISA response in relation to vaccination is recommended.


Scalable superconducting nanowire single photon detector (SNSPDs) arrays require cryogenic digital circuits for multiplexing the output detection pulses. Among existing superconducting digital devices, superconducting nanowire cryotron (nTron) is a three-terminal device with an ultra-compact size, which is promising for large scale monolithic integration. In this report, in order to evaluate the potential and possibility of using nTrons for reading and digitizing SNSPD signals, we characterized the grey zone, speed, timing jitter and power dissipation of a proper designed nTron. With a DC bias on the gate, the nTron can be triggered by a few μA high and nanoseconds wide input signal, showing the nTron was capable of reading an SNSPD pulse at the same signal level. The timing jitter depended on the input signal level. For a 20 μA high and 5 ns wide input pulse, the timing jitter was 33.3 ps, while a typical SNSPD’s jitter was around 50 ps. With removing the serial inductors and operating it in an AC bias mode. The nTron was demonstrated to be operated at a clock frequency of 615.4 MHz, which was faster than the maximum counting rate of a typical SNSPD. In additional, with a 50 Ω bias resistor and biased at 17.6 μA, the nTron had a total power dissipation of 19.7 nW. Although RSFQ circuits are faster than nTrons, for reading SNSPD or other detector arrays that demands less operation speed, our results suggest a digital circuit made from nTrons could be another promising alternative. It has several advantages, such as ultra-compact size, large fan-out number, and insensitiveness to magnetic noise. In particular, an nTron has large output impedance and can drive a CMOS transistor directly2. It also has been demonstrated that an SFQ pulse can trigger an nTron, so does the output from a superconducting nanowire single-photon detector (SNSPD)2. Besides, nTrons have been used in some hybrid cryogenic circuits, such as driving a CMOS memory3, and driving LEDs in a photonic neuromorphic computer4.Superconducting nanowire cryotron (nTron) is a three-terminal device made from superconducting nanowires and it is capable of building superconducting digital circuits5. Moreover, in an SFQ circuit, any input signal has to be digitalized to SFQ pulses before logic processing, and any output SFQ pulse has to be amplified to a level that room temperature electronics can read. For a superconducting computer, this signal incompatibility is not a severe problem since the amount of digital processing is heavy and most signals could be exchanged inside an SFQ chip. However, for other applications where non-SFQ signals are massively received and the amount of digital processing is moderate, for instance, a digital readout circuit for a superconducting detector array, signal-conversion circuits use much resources7.The interfacing performance of the nTrons is well utilized, suggesting that nTrons are promising in reading signals from superconducting detectors. Although a RSFQ can have clock frequency up to 100 GHz, most superconducting detectors are much slower. Thus, the speed of SFQ circuits are not fully taken advantage of. For example, the SNSPD is single photon counting detector and its counting rate is typically less than 100 million counts per second10, a focal plane detector array integrated with a compact and low power dissipation cryognic readout circuit would be a solution for achieving large scale SNSPD arrays. Since an nTron is also a superconducting nanowire device, it uses the same fabrication processes and can be integrated on a single chip with SNSPDs. Moreover, the switching area of an nTron is confined in a choke so that the integration with an nTron readout circuit can be very compact.Therefore, although nTrons are relatively slower than RSFQ circuits, we think they are promising in reading superconducting detectors, in particular for SNSPDs. Recently, developing large SNSPD arrays is a challenge, where a scalable and power efficient cryogenic signal readout is the key technology. Although some multiplexing readout schemes based on time domain or frequency domain have been demonstrated for reading moderate sized SNSPD arraysIn this report, we proposed an nTron geometry and characterized its grey zone, speed, timing jitter and power dissipation. The experimental results are compared with the signal requirements of a typical SNSPD for evaluating the potential and possibility of using nTrons for reading and digitizing SNSPD signals. We also discussed tradeoffs between these parameters, from which the scalability, cooling budget, operation speed, etc. of a potential nTron digital circuit could be derived.The nTrons were patterned from a ~10 nm-thick niobium nitride (NbN) film deposited on a thermal oxide silicon substrate. As shown in Fig. In this report, we characterized two nTron devices. Both of them used the same geometry. But one nTron had serial bias inductors and the other one had not. For the nTron used in grey zone measurements, the corresponding critical currents were 8.6 μA and 52.0 μA measured at liquid helium temperature. The meandered nanowires connected in series on the gate bias port and channel bias port acted as choke inductors to separate DC bias and RF output. Each bias port was connected in series with a 17.2 kΩ off-chip resistor. In the pulse measurements, we used an nTron without the serial inductors to minimize the overall inductance and replaced the bias resistors by 50 Ω resistors so that fast pulses can be applied with mimimum reflections. The fast nTron was fabricated on a thinner film. Therefore, the gate critical current reduced to 6.0 μA and the channel critical current was 21.9 μA. The input port and the output port each connected in series with an external 50 Ω resistor on a PCB to match the impedance of 50 Ω coaxial cables.A circuit schematic diagram of the nTron is shown in Fig. Igh defines the lower level for the input logic 1, while the maximum amplitude of the input current pulse for not triggering an nTron channel IgL defines the upper level for the input logic 0. Between IgL and IgH, there is an unstable region where an nTron switching stochastically due to thermal and quantum fluctuations11. This unstable region is referred to as grey zone12. Figure tw varying from 5 ns to 1.2 μs. For each point, we sent Nin = 5 × 105 pulses to the gate and counted the number of switched pulses Ntr. The switching probability was defined as Ntr/Nin. The channel bias was p = 0.5 as the input threshold currents Igth. The grey zone of GZ was defined as GZ = 1/ρ where ρ was the slope of the switching probability curve at the input pulse amplitude equal to Igth.An nTron operates like a digital comparator when it is used to read out SNSPD pulses. Since an nTron has current gain, it will amplify the SNSPD pulse into a large output pulse. We can take SNSPD pulse as digital input as well. When nTrons are used in building digital circuits, the switching behavior determines the logic levels and bias margin. As shown in Fig. Igth and GZ on input pulse width. As shown in Fig. Igth. As an nTron channel’s output impedance was much higher than a typical 50 Ω load, the output current was proportional to its initial bias. With ignoring the leakage current through the channel, we can have the current gain of the nTron, which was tw = 5 ns, corresponding to a frequency of 200 MHz, G was 13.0. With a wider pulse, for instance, 1/tw = 833 kHz, G increased to 23.9. We noticed that the GZ had a similar dependence on tw. Because there was noise adding on input pulses, when a wider pulse was applied, it was more likely to trigger an nTron, resulting in a narrower transition of the switching probability curve and a lower GZ value. Similar to what have been done on JJs, we can model the nTron switching dynamics with taking into account an energy barrier for escaping11.With data shown in Fig. 14. Thus, an nTron should have intrinsic low timing jitter so that the low timing jitter of an SNSPD read after an nTron digital circuit can be maintained. To study the switching jitter of the nTron shown in Fig. 4 pulses were collected by a high-speed oscilloscope for calculating the statistic values.Low timing jitter is one of SNSPD’s advantages. Depending on the geometry, output signal-to-noise ratio, biasing current et. al, the timing jitter defined at full-width-at-half-magnitude varies from 3 ps to ~50 psFigure 1, Adam McCaughan and Karl Berggren suggested a 2D electro-thermal model, which could be used to simulate the switching dynamics for nTrons of different geometries. Since the nTron gate was 20 nm while the coherent length of NbN was about 10 nm, phase slippage and vortex dynamics in a nanowire can happen before a stable resistive domain was generated, which could be the sources for introducing wider grey zone and larger time jitter15. Based on the experimental results we have now, we cannot conclude a precise switching model for nTrons. However, the measured current threshold and timing jitter of the proposed nTron geometry can meet the requirement for reading an SNSPD-like pulse, i.e. 5 ns wide and 20 μA high.Both the timing jitter and the grey zone measurements indicated that the switching of an nTron started from probabilistic to deterministic as the input pulse increased. To understand such behavior of nTron needed a switching dynamic model with taking account fluctuations. In the first paper of nTron published in ref.16. To reduce the electric recovery time, we removed the serial inductors on the bias ports, and shortened the nanowire to have less inductance. When nTrons were integrated on a single chip, connection length can be further reduced to speed up the electric recovery.The grey zone measurements showed that the nTron can be triggered by nanoseconds wide pulses, implying a possible operation speed of hundreds of MHz. To have a better characterization of the operation speed, i.e. the maximum clock frequency, of an nTron digital circuit, we measured the recovery time of a fast nTron. As nTrons were made from NbN nanowires, which had high kinetic inductance, current charging and discharging caused an electrical recovery timeAnother recovery time was from thermal relaxation of the local hotspot generated on a fired nTron. The hotspot was a resistive area where the local Joule heating increased the hotspot temperature. The power of input pulse can control the hotspot size and temperature. Thus, there was a tradeoff between the input signal power and the recovery time (i.e. the speed of nTron).t were sent to the nTron channel. The first bias pulse was 31.0 ns wide and 17.6 μA high. A gate pulse, which was 625 ps wide, was sent to trigger the nTron at the first bias pulse. We aligned the bias pulse and the input pulse to make them drop to zero simultaneously. Then, we sent a second channel bias pulse and swept its amplitude to find the switching current of the nTron channel of t. As we connected a 50 Ω resistor to the nTron gate to match the impedance of the coaxial cable, the input power sending to the nTron was R = 50 Ω. Before the gate nanowire switched, the input power was dissipated mostly on the resistor. Once the gate nanowire switched into a normal resistor, the gate impedance increased much higher than 50 Ω. Thus, most power was dissipated on the nTron gate. Although some power was reflected, making the measurement of actual power dissipation of the gate nanowire difficult, by adjusting Pin we can control the maximum temperature that the gate nanowire reached after a switch, as shown in Fig. To measure the thermal recovery time of an nTron, we implemented a pump-probe like measurement. As shown in Fig. Tch(Δt) by solving Eq.  calculated by  the number of nTron output pulses divided by the total input pulse number was less than 10−4. We swept Ig and measured the bit error rate (BER) of the nTron output. As shown in Fig. Ig for correct operations reduced to ~1 μA although the operation speed increased to 615.4 MHz and the gate input power was Pg = 4.2 nW at Ig = 9.2 μA. The channel was biased at 17.6 uA through a 50 Ω resistor. Thus, the input power to the channel wire was 15.5 nW. The total power sending to the nTron was 19.7 nW.From the above analyses, there was a tradeoff between the operation speed and the input power dissipation. If the input signal was high, although an nTron can be switched deterministically with low timing jitter, the nTron was heated up and needed a longer time for cooling, limiting the operation speed. If input pulses came with an interval time shorter than the relaxation time or the channel current recharged faster than the relaxation time, an nTron would latch. As shown in Fig. 18. The nTron’s power dissipation is better than a cryogenic CMOS transistor, which usually dissipated mW power19. Thus, a mW cooling power could afford ~50 thousand nTrons, carrying out many digital circuits, e.g. a binary-coded decimal encoder or a counter.In conclusion, we investigated the threshold currents, grey zone, speed and power dissipation of a typical nTron made from NbN films and measured at 4.2 K. We found those parameters related closely to each other and tradeoffs between them occurred. Experimentally, we demonstrated an nTron can be operated at a speed of 615.4 MHz with an input gate current of 9.2 μA and a total input power of 19.7 nW. The switching timing jitter of an nTron, included the measurement jitter caused by voltage noise, was 33.3 ps at a 5 ns wide and 20.6 μA high input pulse. Although the nTron’s performance are less superior to JJs in an advanced RSFQ circuit, the nTron we characterized in this work is faster enough to read a relatively slow superconducting detector, for instance an SNSPD that usually have a maximum counting rate less than 100 McpsIt is worth to notice that the performance of an nTron depends on their design and geometry. If the size of the gate is much smaller, the gate critical current reduces so that the gate can be triggered at a low input current. Then, as the gate is narrow and thin, the local temperature of gate is higher with the same power dissipation on the gate. Meanwhile, an nTron made on substrates of high thermal conductivity, i.e. sapphire and MgO, will exhibit faster operation speed due to the short thermal recovery time. An nTron of wider channel can output more current, but the threshold current will increase as well. Understanding those dependences needs to study non-equilibrium dynamics of a superconducting nanowire, which is out of the scope of this work. Once the design of an nTron is made, characterization methods presented in work can be used for extracting its switching performance.22. This could give an nTron a considerable improvement in speed and power dissipation. However, as we mentioned in the introduction, an nTron operated in hotspot regime has its own advantages and is promising in certain digital applications. We wish the measured characteristics and tradeoffs shown in this letter can help researchers to design and utilize nTrons properly.Recent works demonstrate that an nTron can be operated in a flux regime similar to shunted Josephson Junctions if we prevent the nanowire from heating to a hotpot


Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE. Among the several identified endonucleases, including apoptosis-inducing factor (AIF) and EndoG, DNA fragmentation factor 40/45 (DFF40/45) is considered as the main player in the process of DNA fragmentation during apoptosis. Additionally, AIF and EndoG were shown to be involved in initial large-scale DNA fragmentation during apoptosis, while the DFF40/45 hetero-dimeric complex is involved in further small-scale cleavage5. DFF40 is an endonuclease directly involved in the DNA cleavage process, while DFF45 is an inhibitor that suppresses the nuclease activity of DFF40 under normal conditions via a tight interaction6. During the apoptosis signalling process, activated effector caspases, such as caspase-3, cleave DFF45, allow DFF40 to dissociate from DFF45, enter the nucleus, and degrade chromosomal DNA8.The activation of endonucleases with cleavage of chromosomal DNA into ~180-base pair fragments is one of the main features of apoptotic cells9. According to the sequence homology search, CIDE domain-containing proteins such as CIDE-A, CIDE-B, and CIDE-3 (FSP27 in mouse) have been discovered in human genome11. Although the main function of CIDE domain-containing proteins was a regulator of apoptosis, several knock-out mouse studies revealed that they do function on lipid metabolism by localizing to lipid droplets and the endoplasmic reticulum15.Both DFF40 and DFF45 have a common protein interacting module known as the cell death-inducing DFF45-like effect domain (CIDE domain), which is approximately 90 amino acid residuesDrosophila Melanogaster, and DREP1, DREP2, DREP3, and DREP4 have been identified as CIDE domain-containing proteins in fly17. DREP1 and DREP4 are homologues of DFF45 and DFF40, respectively. DREP2 has been suggested as another endonuclease that is inhibited by DREP3 through interactions with CIDE domains20. A recent biochemical study showed that DREP2 interacts with both DREP1 and DREP3 via the CIDE domain21. Novel functions for DREP2 in learning and memory in fly brain synapses have been suggested22. However, the function and biological implications of the interactions of DREP2 with their binding partners in the cell are unknown.Apoptotic DNA fragmentation and lipid metabolism are conserved in 29. We recently reported the filament-like CIDE domain assembly of DFF40 and DREP4 and its functional implications in the apoptotic DNA fragmentation process30. In the initial structural study of CIDE domain assembly, we solved the structure of the DREP2 CIDE domain. However, this structure was not helical in the asymmetric unit, making it unclear how these proteins assemble into highly oligomeric forms in solution. Based on the structural study of DREP4 CIDE, which showed a helical filament-like structure even in the crystallographic asymmetric unit, we found that the CIDE domains of both DREP4 and DREP2 form filament-like structures in solution. In this study, the details of the helical assembly of the CIDE domain were determined and the filament-like helical oligomeric complex of DREP2 CIDE was confirmed by structural analysis and mutagenesis studies. Additionally, the differences in the characteristics of filament-like structures between DREP2 and DREP4 were determined.CIDE domain-mediated protein interactions have been investigated in structural studies of several CIDE domains and hetero-dimeric and homo-dimeric complex structuresApoptotic DNA fragmentation, which is mainly mediated by the DFF40/DFF45 heterocomplex, is the hallmark of apoptotic cell death and conserved in fly. Four CIDE domain-containing proteins have been identified in fly: DREP1, DREP2, DREP3, and DREP4. Unlike DFF45, which contains only conserved acidic residues, the DREP2, DREP4, DFF40, and FSP27 CIDE domains contain two patches, acidic and basic, in one CIDE domain Fig. . Althoug30. To analyse the salt dependency of complex formation of DREP2, we conducted multi-angle light scattering (MALS), which reveals the absolute molecular weight of a particle. The molecular weight of DREP2 CIDE in 50 mM NaCl and 1 M NaCl were calculated by MALS. The theoretical molecular weight of DREP2 CIDE was 10.99 kDa, while the experimental weights determined by MALS were 281.6 kDa (2.04% fitting error) in 50 mM NaCl and 215.3 kDa (0.77% fitting error) in 1 M NaCl, indicating that complex formation of DREP2 CIDE is not dependent on the salt concentration  method followed by refining to an Rand Fig. . This caand Fig. . Unlike and Fig. . A modeland Fig. . Each moand Fig. .Table 1C2 accessible surface area. Inspection of interface C revealed that the main interactions are mediated by polar residues involving K9, K13, W15, R22, K23, and N72 from chain D and D56, T58, Q59, E61, E64, and Y65 from chain C. These residues form three salts bridges and four hydrogen bonds, as well as contribute to van der Waals interactions  and E64 (chain C)  and between chains A and C . Interface B buries the 368-Å2 accessible surface area, and the interaction is mediated by residues E31, T29, and G61 from chain B and E31, K38, and A45 from chain C  showed the most extensive interactions burying the 500-Åons Fig. . The sec C) Fig. . Interesn C Fig. . The lasn A Fig. . In the 29. The solution structure of the hetero-dimeric complex between DFF40 CIDE and DFF45 CIDE showed that the interaction is mediated by a basic patch  on DFF40 CIDE and acidic patch  on DFF45 CIDE27 , which is aligned with K9 in DFF40, to disrupt the interfaces A and B in the tetrameric DREP2 structure. We also introduced a mutation at R36 in the DREP2 CIDE domain to E , which is critical for the formation of interface D in the tetrameric DREP2 structure Fig. . While wwork of 18.0% and Rfree of 23.4%. Four molecules in the asymmetric unit were the same as those in the wild-type  β strands, the high-resolution structure revealed a possible biologically important higher oligomerization mechanism of the CIDE domain that functions through several novel dimeric interfaces formed between homo-dimers. Before solving helical filament-like oligomeric structure of DREP4, determining the DREP2 structure, which formed an unusual tetramer without any symmetry in the crystallographic asymmetry unit, was difficult. The current study showed that DREP2 CIDE also formed a filament-like structure with features differing from those of DREP4 CIDE in solution. The filament-like oligomeric structure of DREP2 was confirmed by mutagenesis analysis.Although the function of CIDE domain-mediated filament-like structure in apoptotic DNA fragmentation was established in the current structural study, CIDE domain-mediated lipid metabolism and learning/memory in brain synapses require further investigation. The characterization and elucidation of the oligomeric forms of the CIDE domain and CIDE domain-containing proteins will provide important information regarding the function of various CIDE domain-containing proteins in apoptosis, lipid metabolism, and particularly lipid droplet (LD) growth and learning/memory in the brain synapses.http://www.ebi.ac.kr/Tools/clustalw2/index.html).Clustal W has been used for analysing the amino acid sequences of CIDEs , the DREP4 CIDE (amino acids 39–130), and FSP27 (amino acids 38–119) were expressed in Escherichia coli BL21 (DE 3) under overnight induction at 20 °C. The protein contained a carboxyl terminal His-tag and was purified by nickel affinity and size-exclusion chromatography with a S200 gel filtration column 10/30  that had been pre-equilibrated with 20 mM Tris-HCl at pH 8.0 and 150 mM or 500 mM NaCl. The protein was then concentrated for further use.The expression and purification methods used in this study have been described in detail elsewhereOligomeric forms of each CIDE domains were monitored by native (non-denaturing) PAGE on a PhastSystem  with pre-made 8–25% acrylamide gradient gels . Separately purified proteins were directly loaded onto the gel. Coomassie Brilliant Blue was used for staining and detection of the shifted bands. The uncropped scan of gel is provided at Supplementary Fig. 33. The mutant crystal was obtained in the similar crystallization condition. 2.3 Å and 3.3 Å data were collected for wildtype and mutant, respectively.The crystallization conditions were initially screened at 20 °C by the hanging drop vapor-diffusion method using various screening kits. Initial crystals were grown on the plates by equilibrating a mixture containing 1 μL of protein solution  and 1 μL of a reservoir solution containing 300 mM magnesium formate dihydrate and 100 mM Bis-Tris pH 6.2 against 0.4 mL of reservoir solution. The best crystal was obtained by further optimization searching over a range of concentrations of protein and precipitant and pH ranges. The diffraction data set was collected at beamline BL-4A of the Pohang Accelerator Laboratory, Republic of Korea. Data processing and scaling were carried out using the HKL2000 package34 with the solution structure of the CIDE-N domain of human cell death activator CIDE-A (PDB code 2EEL), which has 37% amino-acid sequence identity, as a search model. The MR solution gave rotation-function and translation function Z-scores of 5.4 and 18.9, respectively. Model building and refinement were performed using COOT35 and Refmac536, respectively. Model quality was evaluated using PROCHECK37. Pymol was used to generate all the cartoon figures38.The initial molecular-replacement (MR) method was carried out using PhaserSite-directed mutagenesis was conducted using a Quick-change kit  according to the manufacturer’s protocols. Mutagenesis was then confirmed by sequencing. Mutant proteins were prepared as described above.For gel filtration analysis to detect oligomerization formation, the target protein was applied to a gel-filtration column  that had been pre-equilibrated with 20 mM Tris-HCl 8.0 and 500 mM NaCl. The peak fractions were collected and subjected to SDS-PAGE.The molar mass of the highly oligomerized CIDE domain of DREP2 was determined by MALS. The target protein was injected onto a Superdex 200 HR 10/30 gel filtration column . The chromatography system was coupled to a three-angle light scattering detector (mini-DAWN EOS) and refractive index detector (Optilab DSP) . Data were collected every 0.5 s at a flow rate of 0.2 mL/min and analysed using the ASTRA program, which gave the molar mass and mass distribution (polydispersity) of the sample.39.The secondary structures were measured by circular dichroism (CD) spectroscopy using a J-715 spectropolarimeter  at the Korea Basic Science Institute in South Korea. The spectra were obtained from 200 to 250 nm at 25 °C in a 0.1-cm path length quartz cuvette at a bandwidth of 1.0 nm, rate of 50 mm/min, and 5-s response time. The protein samples in buffer containing 20 mM Tris-HCl at pH 8.0 and 150 mM NaCl were diluted to 0.1 mg/mL prior to use. Four scans were accumulated and averaged, after which the α-helical content was calculated from the molar ellipticity at 222 nmWild-type DREP2 CIDE and R36E mutant samples after affinity chromatography purification were diluted to 0.8 mg/mL to prepare the high concentration sample and 0.1 mg/mL to prepare the low concentration sample. DREP4 CIDE samples were also diluted to 1 mg/mL as the high concentration sample and 0.1 mg/mL as the low concentration sample. For negative staining, 10 μL of each protein sample was placed onto a glow discharged copper grid and stained with 1% uranyl formate at pH 4.5 for 30 s and air-dried. The grids were imaged using a Tecnai G² Spirit BioTWIN Transmission Electron Microscope  and recorded with an AMT 2k CCD camera .Coordinates and structural factors have been deposited in the Protein Data Bank.Supplementary Figure