Performance analysis of versatile video coding for encoding phase-only hologram videos.

In recent decades, holographic technology has made significant progress with the development of novel hologram generation methods and three-dimensional rendering devices. Nevertheless, the substantial size of holographic data presents a significant challenge to its practical applications and thus necessitates the implementation of an efficient coding solution. In this study, we evaluate the efficiency of various coding tools within the state-of-the-art video coding standard, Versatile Video Coding, for encoding video of computer-generated phase-only hologram. Specifically, we examine the coding performance of transform/in-loop filter/screen-content coding tools. Through extensive encoding experiments and various statistical analyses, we investigated the limitations of existing standard codecs that do not account for the unique signal characteristics of phase-only holograms (POHs). The effects of coding artifacts on the visual quality of numerical reconstructions rendered from compressed POHs are also analyzed in detail. These comprehensive performance evaluations will provide valuable insights for developing efficient coding strategies for POH videos.

Deep compression network for enhancing numerical reconstruction quality of full-complex holograms.

The field of digital holography has been significant developed in recent decades, however, the commercialization of digital holograms is still hindered by the issue of large data sizes. Due to the complex signal characteristics of digital holograms, which are of interferometric nature, traditional codecs are not able to provide satisfactory coding efficiency. Furthermore, in a typical coding scenario, the hologram is encoded and then decoded, leading to a numerical reconstruction via a light wave propagation model. While previous researches have mainly focused on the quality of the decoded hologram, it is the numerical reconstruction that is visible to the viewer, and thus, its quality must also be taken into consideration when designing a coding solution. In this study, the coding performances of existing compression standards, JPEG2000 and HEVC-Intra, are evaluated on a set of digital holograms, then the limitations of these standards are analyzed. Subsequently, we propose a deep learning-based compression network for full-complex holograms that demonstrates superior coding performance when compared to the latest standard codecs such as VVC and JPEG-XL, in addition to JPEG2000 and HEVC. The proposed network incorporates not only the quality of the decoded hologram, but also the quality of the numerical reconstruction as distortion costs for network training. The experimental results validate that the proposed network provides superior objective coding efficiency and better visual quality compared to the existing methods.

HEVC extension for phase hologram compression.

Compressing digital holograms have growing attention nowadays due to their huge amount of original data sizes. Although many progresses have been reported for full-complex holograms, the coding performance for phase-only holograms (POHs) has been quite limited so far. In this paper, we present a very efficient compression method for POHs. It extends the conventional video coding standard HEVC (High Efficiency Video Coding) in such a way that the standard can be able to compress not only the natural images but also the phase images effectively. First, we suggest a proper way to calculate differences, distances and clipped values for phase signals by considering the inherent periodicity of phases. Then, some of the HEVC encoding and decoding processes are modified accordingly. The experimental results show that the proposed extension significantly outperforms the original HEVC for POH video sequences; specifically, average BD-rate reductions of 63.3% and 65.5% are achieved in phase domain and numerical reconstruction domain, respectively. It is worth mentioning that the modified encoding & decoding processes are very minimal and also applicable to the VVC (Versatile Video Coding), which is a successor of the HEVC.

Video Quality Model of Compression, Resolution and Frame Rate Adaptation Based on Space-Time Regularities.

Being able to accurately predict the visual quality of videos subjected to various combinations of dimension reduction protocols is of high interest to the streaming video industry, given rapid increases in frame resolutions and frame rates. In this direction, we have developed a video quality predictor that is sensitive to spatial, temporal, or space-time subsampling combined with compression. Our predictor is based on new models of space-time natural video statistics (NVS). Specifically, we model the statistics of divisively normalized difference between neighboring frames that are relatively displaced. In an extensive empirical study, we found that those paths of space-time displaced frame differences that provide maximal regularity against our NVS model generally align best with motion trajectories. Motivated by this, we built a new video quality prediction engine that extracts NVS features that represent how space-time directional regularities are disturbed by space-time distortions. Based on parametric models of these regularities, we compute features that are used to train a regressor that can accurately predict perceptual quality. As a stringent test of the new model, we apply it to the difficult problem of predicting the quality of videos subjected not only to compression, but also to downsampling in space and/or time. We show that the new quality model achieves state-of-the-art (SOTA) prediction performance on the new ETRI-LIVE Space-Time Subsampled Video Quality (STSVQ) and also on the AVT-VQDB-UHD-1 database.

A Subjective and Objective Study of Space-Time Subsampled Video Quality.

Video dimensions are continuously increasing to provide more realistic and immersive experiences to global streaming and social media viewers. However, increments in video parameters such as spatial resolution and frame rate are inevitably associated with larger data volumes. Transmitting increasingly voluminous videos through limited bandwidth networks in a perceptually optimal way is a current challenge affecting billions of viewers. One recent practice adopted by video service providers is space-time resolution adaptation in conjunction with video compression. Consequently, it is important to understand how different levels of space-time subsampling and compression affect the perceptual quality of videos. Towards making progress in this direction, we constructed a large new resource, called the ETRI-LIVE Space-Time Subsampled Video Quality (ETRI-LIVE STSVQ) database, containing 437 videos generated by applying various levels of combined space-time subsampling and video compression on 15 diverse video contents. We also conducted a large-scale human study on the new dataset, collecting about 15,000 subjective judgments of video quality. We provide a rate-distortion analysis of the collected subjective scores, enabling us to investigate the perceptual impact of space-time subsampling at different bit rates. We also evaluated and compare the performance of leading video quality models on the new database. The new ETRI-LIVE STSVQ database is being made freely available at (https://live.ece.utexas.edu/research/ETRI-LIVE_STSVQ/index.html).

On the space-time statistics of motion pictures.

It is well known that natural images possess statistical regularities that can be captured by bandpass decomposition and divisive normalization processes that approximate early neural processing in the human visual system. We expand on these studies and present new findings on the properties of space-time natural statistics that are inherent in motion pictures. Our model relies on the concept of temporal bandpass (e.g., lag) filtering in lateral geniculate nucleus (LGN) and area V1, which is similar to smoothed frame differencing of video frames. Specifically, we model the statistics of the differences between adjacent or neighboring video frames that have been slightly spatially displaced relative to one another. We find that when these space-time differences are further subjected to locally pooled divisive normalization, statistical regularities (or lack thereof) arise that depend on the local motion trajectory. We find that bandpass and divisively normalized frame differences that are displaced along the motion direction exhibit stronger statistical regularities than for other displacements. Conversely, the direction-dependent regularities of displaced frame differences can be used to estimate the image motion (optical flow) by finding the space-time displacement paths that best preserve statistical regularity.

Psychometric Properties of the Nine-Item Korean Internet Gaming Disorder Scale: Short Form.

The main objective of this study was to examine the psychometric properties of the Korean version of the Internet Gaming Disorder Scale-Short Form (K-IGDS9-SF), a brief self-report instrument developed to assess Internet gaming disorder (IGD) in accordance with the Diagnostic and Statistical Manual of Mental Disorders-5th edition criteria. A total of 594 Korean game users (average age = 23.5 years, 70.37 percent male) participated and completed questionnaires containing the K-IGDS9-SF and other measures for its validation. The K-IGDS9-SF showed good reliability (Cronbach's α = 0.86, composite reliability coefficient = 0.87). Consistent with previous studies, the K-IGDS9-SF demonstrated a single-factor structure in a confirmatory factor analysis. Moreover, the K-IGDS9-SF had significant associations with related variables (IGD, game playing variables, self-esteem, impulsivity, and loneliness) in the expected direction, supporting its concurrent, criterion, and convergent validity. Taken together, these results indicated that the K-IGDS9-SF has satisfactory psychometric properties, suggesting its utility as a unified robust instrument for studying IGD worldwide.

Quality Prediction on Deep Generative Images.

In recent years, deep neural networks have been utilized in a wide variety of applications including image generation. In particular, generative adversarial networks (GANs) are able to produce highly realistic pictures as part of tasks such as image compression. As with standard compression, it is desirable to be able to automatically assess the perceptual quality of generative images to monitor and control the encode process. However, existing image quality algorithms are ineffective on GAN generated content, especially on textured regions and at high compressions. Here we propose a new "naturalness"-based image quality predictor for generative images. Our new GAN picture quality predictor is built using a multi-stage parallel boosting system based on structural similarity features and measurements of statistical similarity. To enable model development and testing, we also constructed a subjective GAN image quality database containing (distorted) GAN images and collected human opinions of them. Our experimental results indicate that our proposed GAN IQA model delivers superior quality predictions on the generative image datasets, as well as on traditional image quality datasets.

Learning-Based Just-Noticeable-Quantization- Distortion Modeling for Perceptual Video Coding.

Conventional predictive video coding-based approaches are reaching the limit of their potential coding efficiency improvements, because of severely increasing computation complexity. As an alternative approach, perceptual video coding (PVC) has attempted to achieve high coding efficiency by eliminating perceptual redundancy, using just-noticeable-distortion (JND) directed PVC. The previous JNDs were modeled by adding white Gaussian noise or specific signal patterns into the original images, which were not appropriate in finding JND thresholds due to distortion with energy reduction. In this paper, we present a novel discrete cosine transform-based energy-reduced JND model, called ERJND, that is more suitable for JND-based PVC schemes. Then, the proposed ERJND model is extended to two learning-based just-noticeable-quantization-distortion (JNQD) models as preprocessing that can be applied for perceptual video coding. The two JNQD models can automatically adjust JND levels based on given quantization step sizes. One of the two JNQD models, called LR-JNQD, is based on linear regression and determines the model parameter for JNQD based on extracted handcraft features. The other JNQD model is based on a convolution neural network (CNN), called CNN-JNQD. To our best knowledge, our paper is the first approach to automatically adjust JND levels according to quantization step sizes for preprocessing the input to video encoders. In experiments, both the LR-JNQD and CNN-JNQD models were applied to high efficiency video coding (HEVC) and yielded maximum (average) bitrate reductions of 38.51% (10.38%) and 67.88% (24.91%), respectively, with little subjective video quality degradation, compared with the input without preprocessing applied.

Sorafenib controls the epithelial­mesenchymal transition of ovarian cancer cells via EGF and the CD44­HA signaling pathway in a cell type­dependent manner.

Cluster of differentiation (CD) 44 and epidermal growth factor (EGF) are closely involved in cellular migration and have been used as stem cell markers. Although the hyaluronan (HA)­binding CD44 is responsible for enhanced cellular motility, the mechanism underlying its actions in various cell types and clinical conditions have yet to be elucidated. In the present study, the multikinase inhibitor sorafenib was used to investigate the diverse effects of EGF stimulation on epithelial­mesenchymal transition (EMT) in ovarian cancer cells using immunoblotting and reverse transcription­polymerase chain reaction. In addition, the association between EGF and CD44/HA signaling pathways in the control of mesenchymal phenotype was determined by gene silencing with small interfering RNA transfection. EGF stimulation of ovarian cancer cells increased cellular migration, mesenchymal transition, CD44 expression and the activation of matrix metalloproteinase (MMP)­2 and MMP­9. Sorafenib effectively suppressed the loss of epithelial characteristics in EGF­treated SK­OV­3 ovarian cancer cells, via targeting the mitogen­activated protein kinase (MAPK)/extracellular signal­regulated kinase (ERK) pathway. Although treatment of Caov­3 ovarian cancer cells with sorafenib blocked the expression of mesenchymal phenotypes following EGF stimulation, EGF­activated Caov­3 cells exhibited reduced MAPK/ERK signaling. Furthermore, EGF­activated Caov­3 cells increased the expression of hyaluronan synthase 2 and HA­CD44 ligation in EGF­exposed Caov­3 cells, which resulted in the activation of the Ras/Raf/MEK signaling pathway, amplification of migratory activity and the expression of mesenchymal markers, including N­cadherin and vimentin. Furthermore, silencing EGFR in SK­OV­3 cells and CD44 in Caov­3 cells suppressed their migratory activity, through inhibition of the MAPK/ERK pathway. The present results suggested that EGF­mediated signaling may regulate metastasis and invasion of ovarian cancer cells, in a cancer cell type­dependent manner.

Vandetanib and ADAM inhibitors synergistically attenuate the pathological migration of EBV-infected retinal pigment epithelial cells by regulating the VEGF-mediated MAPK pathway.

The extracellular signals induced by vascular endothelial growth factor (VEGF) are implicated in choroidal neovascularization (CNV) and thus, are associated with vision-limiting complications in the human retina. Vandetanib is an oral anticancer drug that selectively inhibits the activities of VEGF receptor and epidermal growth factor receptor tyrosine kinase; however, the effects of vandetanib on VEGF in retinal pigment epithelial (RPE) cells have not yet been studied. In the present study, a combined treatment of vandetanib and a disintegrin and metalloproteinase (ADAM) protein inhibitors were used to assess the regulation of Epstein-Barr virus (EBV)-infected ARPE19 cells (ARPE19/EBV) migration as a model of CNV. Vandetanib suppressed the expression of the mesenchymal markers ADAM10 and ADAM17 in ARPE19/EBV cells, and also upregulated epithelial cell markers of the RPE cells, E-cadherin and N-cadherin. The migratory activity of ARPE19/EBV induced by VEGF was efficiently blocked by vandetanib. Furthermore, co-treatment with vandetanib and an ADAM10 inhibitor (GI254023X) or ADAM17 inhibitor (Marimastat) synergistically prevented migration and the expression of vimentin, Snail and α-smooth muscle actin by regulating extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. These results suggest that a combination treatment of vandetanib and ADAM inhibitors may be developed as a novel therapeutic regimen to control retina neovascular disease.

Structured learning algorithm for detection of nonobstructive and obstructive coronary plaque lesions from computed tomography angiography.

Visual identification of coronary arterial lesion from three-dimensional coronary computed tomography angiography (CTA) remains challenging. We aimed to develop a robust automated algorithm for computer detection of coronary artery lesions by machine learning techniques. A structured learning technique is proposed to detect all coronary arterial lesions with stenosis [Formula: see text]. Our algorithm consists of two stages: (1) two independent base decisions indicating the existence of lesions in each arterial segment and (b) the final decision made by combining the base decisions. One of the base decisions is the support vector machine (SVM) based learning algorithm, which divides each artery into small volume patches and integrates several quantitative geometric and shape features for arterial lesions in each small volume patch by SVM algorithm. The other base decision is the formula-based analytic method. The final decision in the first stage applies SVM-based decision fusion to combine the two base decisions in the second stage. The proposed algorithm was applied to 42 CTA patient datasets, acquired with dual-source CT, where 21 datasets had 45 lesions with stenosis [Formula: see text]. Visual identification of lesions with stenosis [Formula: see text] by three expert readers, using consensus reading, was considered as a reference standard. Our method performed with high sensitivity (93%), specificity (95%), and accuracy (94%), with receiver operator characteristic area under the curve of 0.94. The proposed algorithm shows promising results in the automated detection of obstructive and nonobstructive lesions from CTA.

Suppression of E-cadherin mediates gallotannin induced apoptosis in Hep G2 hepatocelluar carcinoma cells.

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin ß1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or ß-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

Inhibition of protein kinase C α/ßII and activation of c-Jun NH2-terminal kinase mediate glycyrrhetinic acid induced apoptosis in non-small cell lung cancer NCI-H460 cells.

Though glycyrrhetinic acid (GA) from Glycyrrhiza glabra was known to exert antioxidant, antifilarial, hepatoprotective, anti-inflammatory and anti-tumor effects, the antitumor mechanism of GA was not clearly elucidated in non-small cell lung cancer cells (NSCLCCs). Thus, in the present study, the underlying apoptotic mechanism of GA was examined in NCI-H460 NSCLCCs. GA significantly suppressed the viability of NCI-H460 and A549 non-small lung cancer cells. Also, GA significantly increased the sub G1 population by cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells in a concentration dependent manner in NCI-H460 non-small lung cancer cells. Consistently, GA cleaved poly (ADP-ribosyl) polymerase (PARP), caspase 9/3, attenuated the expression of Bcl-XL, Bcl-2, Cyclin D1 and Cyclin E in NCI-H460 cells. Interestingly, GA attenuated the phosphorylation of protein kinase C (PKC) α/ßII and extracellular activated protein kinase (ERK) as well as activated the phosphorylation of PKC δ and c-Jun NH2-terminal kinase in NCI-H460 cells. Conversely, PKC promoter phorbol 12-myristate 13-acetate (PMA) and JNK inhibitor SP600125 reversed the cleavages of caspase 3 and PARP induced by GA in NCI-H460 cells. Overall, our findings suggest that GA induces apoptosis via inhibition of PKC α/ßII and activation of JNK in NCI-H460 non-small lung cancer cells as a potent anticancer candidate for lung cancer treatment.

Regulation of crosstalk between epithelial to mesenchymal transition molecules and MMP-9 mediates the antimetastatic activity of anethole in DU145 prostate cancer cells.

The underlying antimetastatic mechanism of anethole (1) still remains unclear in association with the molecules of the epithelial to mesenchymal transition (EMT). Herein, the role of the EMT molecules was elucidated in terms of the antimetastatic activity of 1 using DU145 cells. Anethole significantly inhibited the adhesion of DU145 cells to vitronectin-coated plates, as well as migration in a wound-healing assay and invasion using a Boyden chamber. Also, anethole suppressed the expression of MMP-9 in DU145 cells by zymography, ELISA, and RT-PCR. Consistently, the silencing of MMP-9 enhanced the activity of 1 to upregulate the expression of E-cadherin and to attenuate the expression of Vimentin in DU145 cells. Compound 1 enhanced E-cadherin, which is an epithelial marker and attenuated the expression of Vimentin, Twist, and Snail as mesenchymal molecules at the mRNA level. Consistently, anethole upregulated E-cadherin and downregulated the expression of Vimentin, Twist and PI3K, and AKT at the protein level in DU145 cells. Conversely, the antimetastatic effects of 1 to inhibit invasion and the expression of MMP-9 and upregulate E-cadherin were reversed by the EMT inducer TGF-ß in DU145 cells. Overall, the present findings suggest that anethole exerts antimetastatic activity via regulation of crosstalk between EMT molecules and MMP-9 on the basis of the in vitro data obtained.

Anti-diabetic potential of the essential oil of Pinus koraiensis leaves toward streptozotocin-treated mice and HIT-T15 pancreatic ß cells.

The metabolic syndrome creates risk factors for coronary heart disease, diabetes, fatty liver, obesity and several cancers. Our group has already reported that the essential oil from leaves of Pinus koraiensis SIEB (EOPK) exerted antihyperlipidemic effects by upregulating the low-density lipoprotein receptor and inhibiting acyl-coenzyme A, cholesterol acyltransferases. We evaluated in the current study the anti-diabetic effects of EOPK on mice with streptozotocin (STZ)-induced type I diabetes and on HIT-T15 pancreatic ß cells. EOPK significantly protected HIT-T15 cells from STZ-induced cytotoxicity and reduced the blood glucose level in STZ-induced diabetic mice when compared with the untreated control. EOPK consistently and significantly suppressed the α-amylase activity in a dose-dependent manner and enhanced the expression of insulin at the mRNA level in STZ-treated HIT-T15 cells, while the expression of insulin was attenuated. EOPK also significantly abrogated the population of reactive oxygen species when compared to the untreated control in STZ-treated HIT-T15 cells. Furthermore, EOPK significantly reduce nitric oxide production, suppressed the phosphorylation of endothelial nitric oxide (NO) synthase and suppressed the production of vascular endothelial growth factor (VEGF) in STZ-treated HIT-T15 cells, implying its potential application to diabetic retinopathy. Overall, our findings suggest that EOPK had hypoglycemic potential by inhibiting reactive oxygene species (ROS), endothelial NO synthase (eNOS) and VEGF in STZ-treated mice and HIT-T15 pancreatic ß cells as a potent anti-diabetic agent.

Anti-nephrolithic potential of resveratrol via inhibition of ROS, MCP-1, hyaluronan and osteopontin in vitro and in vivo.

BACKGROUND: Though resveratrol is known to have anti-cancer, anti-diabetic, anti-oxidant and anti-inflammatory activities, the inhibitory mechanism of resveratrol in kidney stone formation has not been elucidated so far. METHOD: ELISA, flow cytometry, RT-PCR, and western blotting were performed. Human renal epithelial cells (HRCs) and rats with ethylene glycol (EG)-induced kidney stones were used. RESULTS: A wound healing assay revealed that resveratrol significantly inhibited the oxalate-mediated migration of HRCs, considering oxalate mediates kidney stone formation. Also, resveratrol suppressed the mRNA expression of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase subunits such as p22(phox) and p47(phox), monocyte chemoattractant protein 1 (MCP-1) and osteopontin (OPN) in oxalate-treated HRCs. Furthermore, western blotting showed that resveratrol downregulated the expression of MCP-1-related proteins including transforming growth factor(TGF-ß1), TGFR-I or II and hyaluronan in oxalate-treated HRCs. Consistently, resveratrol reduced oxalate-mediated production of reactive oxygen species (ROS) and malondialdehyde (MDA) in oxalate-treated HRCs, while the activities of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were enhanced by resveratrol in HRCs and EG-treated kidneys of rats. Consistently, resveratrol significantly reduced the number of urine calcium oxalate crystals and serum MDA, and attenuated the expression of OPN and hyaluroran in EG-treated rats. CONCLUSIONS: Our findings suggest that resveratrol exerts anti-nephrolithic potential via inhibition of ROS, MCP-1 hyaluronan and OPN signaling.

Essential Oil of Pinus koraiensis Exerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling.

Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPAR γ and CEBP α in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPAR γ during adipocyte differentiation. Furthermore, PPAR γ inhibitor GW9662 enhanced the decreased expression of FABP and PPAR γ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPAR γ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPAR γ -related signaling.

Verification of the interdomain contact site in the inactive monomer, and the domain-swapped fold in the active dimer of Hsp33 in solution.

Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain-swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.

Thermal denaturation of the apo-cyclic AMP receptor protein and noncovalent interactions between its domains.

Cyclic AMP receptor protein (CRP) is allosterically activated by cAMP and functions as a global transcription regulator in enteric bacteria. Structural information on CRP in the absence of cAMP (apo-CRP) is essential to fully understand its allosteric behavior. In this study we demonstrated interdomain interactions in apo-CRP, using a comparative thermodynamic approach to the intact protein and its isolated domains, which were prepared either by limited proteolysis or using recombinant DNA. Thermal denaturation of the intact apo-CRP, monitored by differential scanning calorimetry, revealed an apparently single cooperative transition with a slight asymmetry. Combined with circular dichroism and fluorescence analysis, the thermal denaturation of apo-CRP could be interpreted as a coupled process involving two individual transitions, each attributable to a structural domain. When isolated individually, both of the domains exhibited significantly altered thermal behavior, thus pointing to the existence of non-covalent interdomain interactions in the intact apo-CRP. These observations suggest that the allosteric conformational change of CRP upon binding to cAMP is achieved by perturbing or modifying pre-existing interdomain interactions. They also underline the effectiveness of a comparative approach using calorimetric and structural probes for studying the thermodynamics of a protein.