Two activating phosphorylation sites of Pbs2 MAP2K in the yeast HOG pathway are differentially dephosphorylated by four PP2C phosphatases Ptc1-Ptc4.

To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.

Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K.

The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism.

MCRIP1, an ERK substrate, mediates ERK-induced gene silencing during epithelial-mesenchymal transition by regulating the co-repressor CtBP.

The ERK pathway not only upregulates growth-promoting genes, but also downregulates anti-proliferative and tumor-suppressive genes. In particular, ERK signaling contributes to repression of the E-cadherin gene during epithelial-mesenchymal transition (EMT). The CtBP transcriptional co-repressor is also involved in gene silencing of E-cadherin. However, the functional relationship between ERK signaling and CtBP is unknown. Here, we identified an ERK substrate, designated MCRIP1, which bridges ERK signaling and CtBP-mediated gene silencing. CtBP is recruited to promoter elements of target genes by interacting with the DNA-binding transcriptional repressor ZEB1. We found that MCRIP1 binds to CtBP, thereby competitively inhibiting CtBP-ZEB1 interaction. When phosphorylated by ERK, MCRIP1 dissociates from CtBP, allowing CtBP to interact with ZEB1. In this manner, the CtBP co-repressor complex is recruited to, and silences, the E-cadherin promoter by inducing chromatin modifications. Our findings reveal a molecular mechanism underlying ERK-induced epigenetic gene silencing during EMT and its dysregulation in cancer.

Absolute measurement of sampling jitter in audio equipment.

This study proposes a method for analyzing sampling jitter in audio equipment based on the time-domain analysis, considering the temporal fluctuations of the zero-crossing points in the recorded sinusoidal waves to characterize the jitter. This method enabled the separate evaluation of jitter in an audio player from those in audio recorders when the same playback signal is simultaneously fed into two audio recorders. The experiments were conducted using commercially available portable devices with a maximum sampling rate of 192 000 samples per second. The results demonstrated that jitter values on the order of a few tens of picoseconds can be identified in an audio player. Moreover, the proposed method enabled the separation of jitter from phase-independent noise utilizing the left and right channels of the audio equipment. As such, this method is applicable for performance evaluation of audio equipment, signal generators, and clock sources.

Block-like and cast-like hyperdense areas in the right heart cavities on post-mortem CT strongly suggest the presence of intracardiac blood clots at autopsy.

OBJECTIVE: To classify the types of hyperdense areas in the heart cavities on post-mortem CT (PMCT) and compare them according to the presence of blood clots in the heart cavities at forensic autopsy. METHODS: One hundred and twelve cases with CT images taken before forensic autopsy were evaluated. The presence and shape of hyperdense areas in the right or left heart cavities were retrospectively evaluated on PMCT images and were classified into four types (block-like, cast-like, fluid level-like, and unclear). The presence of blood clots was confirmed when there were clots in the heart cavities at forensic autopsy. RESULTS: Of the 112 cases, 57 exhibited blood clots in the heart cavities at forensic autopsy. The hyperdense areas in the right heart cavities on PMCT in 57 cases exhibiting blood clots at forensic autopsy were classified as follows: block-like, 32; fluid level-like, 4; cast-like, 17; and unclear, 4. The sensitivity of block-like and cast-like hyperdense areas in the right heart cavities on PMCT for the presence of clots in the heart cavities at forensic autopsy was 86% (95% confidence interval [CI]: 74-94%); the corresponding specificity, PPV, and NPV were 95% (95% CI: 85-99%), 94% (95% CI: 84-99%), and 87% (95% CI: 75-94%), respectively. CONCLUSION: Block-like and cast-like hyperdense areas in the right heart cavities on PMCT predicted the presence of intracardiac blood clots at forensic autopsy. KEY POINTS: • Clinical radiologists likely have no experience of interpreting findings of blood clots on post-mortem CT (PMCT). • The appearance of blood clots on PMCT provides important clues for diagnosing the cause and process of death. • The shapes of the hyperdense areas in the heart cavities were classified into four types, and two of these types could be used to predict the presence of blood clots in the heart cavities at forensic autopsy.

[Examination of Optimal Window Size and Acquisition Time of Respiratory-gated PET Image: Phantom Study with a SiPM-based PET/CT Scanner].

PURPOSE: This phantom study aimed to determine the optimal acquisition window size for phase-based respiratory gating in silicon photomultiplier (SiPM)-based fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) and its acquisition time in respiratory-gated imaging with the optimal window size. METHODS: Images of a moving NEMA IEC Body Phantom SetTM with hot spheres were acquired. First, the tumor volume and the maximum standardized uptake value (SUVmax) of images reconstructed using a different window size were evaluated to define the optimal window size. Second, the quality of the images reconstructed using the optimal window size and different acquisition times was evaluated using the detectability score of the 10-mm hot sphere and physical indices. RESULTS: The volume and the SUVmax of the 10-mm hot sphere were improved when the window size was narrow, and there were no significant differences among images reconstructed using a window size narrower than 20%. To reconstruct an image using the 20% window size, an acquisition time of 5 min was required to visualize the 10-mm hot sphere. CONCLUSIONS: The optimal window size for phase-based respiratory gating is 20%. Further, an acquisition time of 5 min should be taken for respiratory-gated imaging with the 20% window size on SiPM-based FDG-PET/CT.

Dynamic control of yeast MAP kinase network by induced association and dissociation between the Ste50 scaffold and the Opy2 membrane anchor.

Membrane localization of the Ste11 MAPKKK is essential for activation of both the filamentous growth/invasive growth (FG/IG) MAP kinase (MAPK) pathway and the SHO1 branch of the osmoregulatory HOG MAPK pathway, and is mediated by binding of the Ste50 scaffold protein to the Opy2 membrane anchor. We found that Opy2 has two major (CR-A and CR-B), and one minor (CR-D), binding sites for Ste50. CR-A binds Ste50 constitutively and can transmit signals to both the Hog1 and Fus3/Kss1 MAPKs. CR-B, in contrast, binds Ste50 only when Opy2 is phosphorylated by Yck1/Yck2 under glucose-rich conditions and transmits the signal preferentially to the Hog1 MAPK. Ste50 phosphorylation by activated Hog1/Fus3/Kss1 MAPKs downregulates the HOG MAPK pathway by dissociating Ste50 from Opy2. Furthermore, Ste50 phosphorylation, together with MAPK-specific protein phosphatases, reduces the basal activity of the HOG and the mating MAPK pathways. Thus, dynamic regulation of Ste50-Opy2 interaction fine-tunes the MAPK signaling network.

Postmortem Computed Tomographic Analysis of Death Caused by Oral Drug Intoxication.

Traditional autopsy has changed little in the past century. In Japan, the rate of forensic autopsy in cases of unusual death is very low. Therefore, multi-slice computed tomography (CT) has been used to obtain imaging data instead of or in addition to autopsy in suspicious forensic cases. In our institute, postmortem multi-slice CT has been performed since 2009, and by 2014 there were over 1,000 cases. Our extensive experience with postmortem CT shows that in many cases of death by drug overdose, stomach contents exhibit high X-ray absorption. This article reviews the relationship between CT findings of stomach contents and toxicological analysis results in 23 cases of death by drug overdose. All cases (12 females and 11 males, aged 44 ± 11 years) known to have orally ingested drugs were included in this study. We assessed the slices of all stomach areas on consecutive axial CT images. Twenty cases (87%) showed high X-ray absorption in the stomach, while the other three did not demonstrate radio-dense stomach contents even though drug analysis detected lethal concentrations of drugs in the blood. In conclusion, drugs were frequently, but not always, visualized as contents with high X-ray absorption in the stomach. Postmortem gastric CT images can provide useful information in cases of oral drug intoxication if there are empty drug packages or a suicide note at the death scene. However, precise determination of the cause of death requires full autopsy in cases where there is no indication of suicide at the death scene.

Motion-frozen Imaging by Gated Myocardial Perfusion Single Photon Emission Computed Tomography Using Multi-focus Fan Beam Collimator in Thallium-201 Study.

PURPOSE: This study aimed to evaluate the statistical noise of motion-frozen (MF) image generated by gated myocardial perfusion single photon emission computed tomography (SPECT) imaging using IQ · SPECT and to determine the optimal acquisition and reconstruction parameters for MF image using IQ · SPECT. METHODS: A movement cardiac phantom and static cardiac phantom were used to acquire the MF images. The acquisition times used were different in 8 and 16 frames per R-R interval, and varying reconstruction parameters (subset and iteration) were used. We determined the %CV value, contrast, and normalized mean square error (NMSE) to evaluate the image quality. RESULTS: The %CV value for a MF image with IQ · SPECT was lower than that for a conventional non-gated myocardial perfusion SPECT (MPS) image with low energy high resolution (LEHR). With regard to the acquisition parameters, the contrast did not change when the acquisition time was increased in 8 and 16 frames per R-R interval. NMSE converged in 56 beats/view in 8 frames per R-R interval. With regard to the reconstruction parameters, the contrast and the %CV value of the anterior and septal wall converged in update 40. The minimum NMSE in subsets 1, 2, and 3 were almost similar. CONCLUSIONS: Uniformity in the MF image with IQ · SPECT was higher than that in the conventional image. The results of this MF image with IQ · SPECT study suggest that the optimal acquisition parameter should be 56 beats/view in 8 frames per R-R interval, and the optimal reconstruction parameters should be subset 3 and iteration 14.

A Case Report of Postmortem Radiography of Acute, Fatal Abdominal Distension After Binge Eating.

This case report describes a woman who developed fatal gastric dilatation after binge eating. She called an ambulance because of stomach pain. When she arrived at the hospital, she did not look seriously ill. However, she rapidly became unconscious and collapsed immediately after she was laid on the examination table in a supine position. Postmortem chest x-ray and computed tomography showed right shift of the mediastinum and raised left diaphragm caused by massive gastric distension. Computed tomography showed no visible inferior vena cava. We think that her sudden deterioration was caused by movement of her stomach contents. Radiographic images provided some clues to the cause of her rapid collapse and death.

Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK.

The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG-specific Kss1 MAPK is activated by a combination of an O-glycosylation defect caused by disruption of the gene encoding the protein O-mannosyltransferase Pmt4, and an N-glycosylation defect induced by tunicamycin. The O-glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG-specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.

Use of copper alloy for preventing transmission of methicillin-resistant Staphylococcus aureus contamination in the dermatology ward.

Metallic copper has been shown significantly to reduce methicillin-resistant Staphylococcus aureus (MRSA) contamination of the ambient surroundings of the beds of MRSA-carrying patients in dermatology wards. The aim of this study was to determine whether a bed sheet made of copper-coated film will reduce the spread of MRSA contamination in the environment of a heavily-colonized patient. The bacterial count was highest on the bed sheet. MRSA cell counts on the surface of the non-film-coated control sheet were high (6,600-11,000 colony forming units (cfu)), but those on the copper film were considerably lower (20-130 cfu). Use of metallic copper on the bed sheets of patients who are likely to be a source of MRSA contamination may help to prevent the spread of MRSA contamination in hospital wards.

Cardiac output obtained from test bolus injections as a factor in contrast injection rate revision of following coronary CT angiography.

BACKGROUND: Optimal contrast enhancement is crucial for the detection of coronary artery stenoses and atherosclerotic changes in coronary CT angiography (CTA). PURPOSE: To demonstrate the feasibility of using the cardiac output (CO) obtained from the test bolus injection data-set (COtest) as a factor in contrast injection rate revision of the following coronary CTA. MATERIAL AND METHODS: The test bolus injection data-sets of 52 consecutive coronary CTAs were examined. COtest was calculated from the test bolus data-set. Aortic peak enhancement (APE) was measured on the following coronary CTA. We simulated the APE at a fixed contrast injection rate of 4 mL/s (simAPE) in each patient. RESULTS: The ranges of COtest and simAPE were 2.82-7.56 L/min and 194-527 Hounsfield Units, respectively. There was a significant negative correlation (R = -0.802, P < 0.001) between simAPE and COtest. CONCLUSION: COtest can be used for injection rate revision on coronary CTA.

Using inverse Laplace transform in positronium lifetime imaging.

Positronium (Ps) lifetime imaging is gaining attention to bring out additional biomedical information from positron emission tomography (PET). The lifetime of Psin vivocan change depending on the physical and chemical environments related to some diseases. Due to the limited sensitivity, Ps lifetime imaging may require merging some voxels for statistical accuracy. This paper presents a method for separating the lifetime components in the voxel to avoid information loss due to averaging. The mathematics for this separation is the inverse Laplace transform (ILT), and the authors examined an iterative numerical ILT algorithm using Tikhonov regularization, namely CONTIN, to discriminate a small lifetime difference due to oxygen saturation. The separability makes it possible to merge voxels without missing critical information on whether they contain abnormally long or short lifetime components. The authors conclude that ILT can compensate for the weaknesses of Ps lifetime imaging and extract the maximum amount of information.

Cervical intervertebral separation caused by trauma on post-mortem computed tomography: Possibility of a diagnosis based on intervertebral gas.

OBJECTIVES: Gas is a common finding in cervical intervertebral separation. However, intervertebral gas is also found in many decedents without intervertebral separation. Here, we quantified intervertebral gas and examined its value in the diagnosis of cervical intervertebral separation. METHODS: We retrospectively reviewed data from 1118 decedents who underwent post-mortem computed tomography (CT) and autopsy from May 2011 to July 2016 and selected those with cervical intervertebral gas with or without intervertebral separation. These data comprised 56 cervical intervertebral spaces with gas [intervertebral separation in 19 (33.9%)] in 43 subjects [intervertebral separation in 17 (39.5%)]. We categorised the decedents according to gas volume, position, and shape and determined the significance of the differences between the decedents with and without separation. RESULTS: The gas volume did not differ significantly between decedents with and without separation (p = 0.063). However, there were significant differences in the gas position between decedents with and without separation. In the sagittal plane: gas was seen in the "centred" position in the ventral-to-dorsal direction in more decedents without separation than in those with separation (p = 0.018). Gas was seen in the ventral-to-dorsal positions in more decedents with separation than in those without separation (p = 0.049). In the cranio-caudal direction, gas in the upper position was more common in decedents with separation than in those without separation in the sagittal plane (p = 0.03). In the coronal plane: gas was seen in the upper position more frequently in decedents with separation in the cranio-caudal direction than in those without separation (p = 0.001). A significant difference in gas shape was observed only in the coronal plane (p = 0.024); irregular gas was associated with decedents without separation. CONCLUSION: Gas in the ventral-to-dorsal and upper positions in the sagittal plane and in the upper position in the coronal plane was rather indicative of cervical intervertebral separation. An irregular gas shape in the coronal plane was indicative of degenerative changes.

Structural basis for the function and regulation of the receptor protein tyrosine phosphatase CD45.

CD45 is the prototypic member of transmembrane receptor-like protein tyrosine phosphatases (RPTPs) and has essential roles in immune functions. The cytoplasmic region of CD45, like many other RPTPs, contains two homologous protein tyrosine phosphatase domains, active domain 1 (D1) and catalytically impaired domain 2 (D2). Here, we report crystal structure of the cytoplasmic D1D2 segment of human CD45 in native and phosphotyrosyl peptide-bound forms. The tertiary structures of D1 and D2 are very similar, but doubly phosphorylated CD3zeta immunoreceptor tyrosine-based activation motif peptide binds only the D1 active site. The D2 "active site" deviates from the other active sites significantly to the extent that excludes any possibility of catalytic activity. The relative orientation of D1 and D2 is very similar to that observed in leukocyte common antigen-related protein with both active sites in an open conformation and is restrained through an extensive network of hydrophobic interactions, hydrogen bonds, and salt bridges. This crystal structure is incompatible with the wedge model previously suggested for CD45 regulation.

Photoexcited triplet states of new UV absorbers, cinnamic acid 2-methylphenyl esters.

Phosphorescence spectra of nonphosphorescent or very weakly phosphorescent new UV absorbers, 2-methylphenyl cinnamate (MePC), 2-methylphenyl 4-methoxycinnamate (MePMC) and 2-methylphenyl 4-ethoxycinnamate (MePEC) have been observed by using external heavy atom effects of ethyl iodide in ethanol at 77 K. The lowest excited triplet (T(1)) energies of these new UV absorbers are lower than those of a widely used UV-A absorber, 4-tert-butyl-4'-methoxydibenzoylmethane (BM-DBM), in both keto and enol forms. The intermolecular triplet-triplet energy transfer from photolabile BM-DBM to MePMC was observed by measuring the time-resolved phosphorescence spectra. Electron paramagnetic resonance spectra have been observed for the T(1) states of these new UV absorbers in ethanol at 77 K by using benzophenone as a triplet sensitizer. The observed T(1) lifetimes, zero-field splitting (ZFS) parameters and molecular orbital calculations of the ZFS parameters suggest that T(1) states of these new UV absorbers posses mainly (3)ππ* character. The deactivation processes of the lowest excited singlet (S(1)) states are predominantly fluorescence and internal conversion to the ground (G) states in MePMC and MePEC, while the main deactivation process of the S(1) state of MePC is internal conversion to the G state. The molar absorption coefficients of MePMC and MePEC in the UV-A and UV-B regions are larger than that of most widely used UV-B absorber, octyl methoxycinnamate.

Accuracy of spectral curves at different phantom sizes and iodine concentrations using dual-source dual-energy computed tomography.

To validate the accuracy of spectral curves obtained by an image-data-based algorithm and clarify the error factors that reduce accuracy. Iodine rods of known composition and different concentrations were inserted into a cylinder or elliptic-cylinder phantom and scanned according to the dual-energy protocol. Spectral curves were obtained by (i) theoretical calculation, (ii) image-data-based 2-material decomposition, and (iii) using a dedicated workstation. Accuracy was verified by comparing the spectral curve obtained by theoretical calculations with those obtained by the image-data-based algorithms or the dedicated workstations. For a quantitative evaluation, the error and relative error (RE) were calculated. In the image-data-based calculation, the errors with respect to the theoretical CT number ranged from - 8.3 to 71.1 HU. For all 192 combinations, 80.7% of the errors were under ± 15 HU, and 97.9% of the REs were under 10%. In the dedicated workstation, the errors ranged from - 94.7 to 26.8 HU. For all combinations, 68.8% of the errors were under ± 15 HU, and 68.2% of the REs were under 10%. By appropriately setting the effective energy corresponding to the CT number of the basis materials, an accurate spectral curve can be obtained. The beam-hardening effect is canceled by the 2-material decomposition process even without beam-hardening correction. Accuracy is primarily reduced by scattered radiation rather than the beam-hardening effect.

Visualization of Cerebrospinal Fluid Motion in the Whole Brain Using Three-dimensional Dynamic Improved Motion-sensitized Driven-equilibrium Steady-state Free Precession.

The feasibility of the 3D dynamic improved motion-sensitized driven-equilibrium steady-state free precession (3D dynamic iMSDE SSFP) was evaluated for visualizing CSF motion and the appropriate parameters were determined. Both flow phantom and volunteer studies revealed that linear ordering and the shortest acquisition duration time were optimal. 3D dynamic iMSDE SSFP provides good quality imaging of CSF motion in the whole brain and enables visualization of flow in arbitrary planes from a single 3D volume scan.

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain.

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.