Advanced Techniques for Liver Fibrosis Detection: Spectral Photoacoustic Imaging and Superpixel Photoacoustic Unmixing Analysis for Collagen Tracking.

Liver fibrosis, a major global health issue, is marked by excessive collagen deposition that impairs liver function. Noninvasive methods for the direct visualization of collagen content are crucial for the early detection and monitoring of fibrosis progression. This study investigates the potential of spectral photoacoustic imaging (sPAI) to monitor collagen development in liver fibrosis. Utilizing a novel data-driven superpixel photoacoustic unmixing (SPAX) framework, we aimed to distinguish collagen presence and evaluate its correlation with fibrosis progression. We employed an established diethylnitrosamine (DEN) model in rats to study liver fibrosis over various time points. Our results revealed a significant correlation between increased collagen photoacoustic signal intensity and advanced fibrosis stages. Collagen abundance maps displayed dynamic changes throughout fibrosis progression. These findings underscore the potential of sPAI for the noninvasive monitoring of collagen dynamics and fibrosis severity assessment. This research advances the development of noninvasive diagnostic tools and personalized management strategies for liver fibrosis.

Brain Targeted Xenon Protects Cerebral Vasculature After Traumatic Brain Injury.

Abstract Cerebrovascular dysfunction following traumatic brain injury (TBI) is a well-characterized phenomenon. Given the therapeutic potential of xenon, we aimed to study its effects after localized delivery to the brain using microbubbles. We designed xenon-containing microbubbles stabilized by dibehenoylphosphatidylcholine (DBPC) and polyethylene glycol (PEG) attached to saturated phospholipid (DPSE-PEG5000). Using a pig model of TBI, these microbubbles were intravenously injected, and ultrasound was used to release xenon at the level of the carotid artery. The control group received perfluorobutane containing microbubbles. Diffusion tensor imaging (DTI) showed areas of higher fractional anisotropy for pigs receiving xenon microbubbles compared to the control group at 1 day after injury. Radial diffusivity analysis showed that this effect was mainly the result of acute edema. Pigs were euthanized at 5 days, and the brain tissues of xenon-treated animals showed reduction of perivascular inflammation and blood-brain barrier disruption. Endothelial cell culture experiments showed that glutamate reduces tight junction protein zona occludens-1 (ZO-1), but treatment with xenon microbubbles attenuates this effect. Xenon treatment protects cerebrovasculature and reduces astroglial reactivity after TBI. Further, these data support the future use of localized delivery of various therapeutic agents for brain injury using microbubbles in order to limit systemic side effects and reduce costs.

Hydralazine-augmented contrast ultrasound imaging improves the detection of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) detection with B-mode and contrast-enhanced ultrasound (CUS) imaging often varies between subjects, especially in patients with background cirrhosis. Various factors contribute to this variability, including the tumor blood flow, tumor size, internal echoes, and its location in livers with diffuse fibro-cirrhotic changes. OBJECTIVE: Towards improving lesion detection, this study evaluates a vasodilator, hydralazine, to enhance the visibility of HCC by reducing its blood flow relative to the surrounding liver tissue. METHODS: HCC were analyzed for tumor visibility measured for B-mode, CUS, and hydralazine-augmented-contrast ultrasound (HyCUS) in an autochthonous HCC rat model. 21 tumors from 12 rats were studied. B-mode and CUS images were acquired before hydralazine injection. Rats received an intravenous hydralazine injection of 5 mg/kg, then images were acquired 20 min later. Four rats were used as controls. The difference in echo intensity of the lesion and the surrounding tissue was used to determine the visibility index (VI). RESULTS: The visibility index for HCC was found to be significantly improved with the use of HyCUS imaging compared to traditional B-mode and CUS imaging. The visibility index for HCC was 16.5 ± 2.8 for HyCUS, compared to 5.3 ± 4.8 for B-mode and 4.1 ± 3.8 for CUS. The differences between HyCUS and the other imaging modalities were statistically significant, with p-values of 0.001 and 0.02, respectively. Additionally, when compared to control cases, HyCUS showed higher discrimination of HCC (VI = 6.4 ± 1.2) with a p-value of 0.003, while B-mode (VI = 6.7 ± 1.4, p = 0.5) and CUS (VI = 6.4 ± 1.2, p = 0.3) showed lower discrimination. CONCLUSION: Vascular blood flow modulation by hydralazine enhances the visibility of HCC. HyCUS offers a potential problem-solving method for detecting HCC when B-mode and CUS are unsuccessful, especially with background fibro-cirrhotic liver disease. Future evaluation of the approach in humans will determine its translatability for clinical applications.

Contrast-Enhanced Ultrasound Tumor Therapy With Abdominal Imaging Transducer.

A diagnostic ultrasound machine add-on module (AOM) was created to enable an off-the-shelf abdominal imaging transducer to perform contrast-enhanced therapeutic ultrasound. The AOM creates plane-wave ultrasound through an abdominal imaging transducer targeting intravascular microbubbles within tumors. This therapeutic antivascular ultrasound (AVUS) causes heating and cavitation effects that destroy tumor vasculature and starves it of nutrients. The AOM can switch between therapeutic and imaging modes for monitoring AVUS treatment. The therapeutic capability of the AOM was validated in murine hepatocellular carcinomas (HCC) grown in adult mice. Contrast-enhanced ultrasound imaging performed before and after the therapeutic treatment evaluated the AVUS response to the treatment. The peak enhancement (PE), perfusion index (PI), and area under the curve (AUC) were measured for the control and AOM treatment groups. The AOM group showed a substantial decrease in these parameters compared to the control group. The difference between the pre- and post-therapy was significant, (p < 0.001) for the AOM group and not significant (p > 0.5) for the control group. Tumor temperatures increased markedly for the AOM group with a thermal dose (CEM43) of 124.8 (±2.5). Histochemical analysis of the excised HCC samples revealed several hemorrhagic pools in tumors from the AOM group, absent in the tumors of the control group. These results demonstrate the theranostic potential of the AOM to induce and monitor vascular disruption within murine tumors.

Ultrasound Radiomics for the Detection of Early-Stage Liver Fibrosis.

Objective: The study evaluates quantitative ultrasound (QUS) texture features with machine learning (ML) to enhance the sensitivity of B-mode ultrasound (US) for the detection of fibrosis at an early stage and distinguish it from advanced fibrosis. Different ML methods were evaluated to determine the best diagnostic model. Methods: 233 B-mode images of liver lobes with early and advanced-stage fibrosis induced in a rat model were analyzed. Sixteen features describing liver texture were measured from regions of interest (ROIs) drawn on B-mode images. The texture features included a first-order statistics run length (RL) and gray-level co-occurrence matrix (GLCM). The features discriminating between early and advanced fibrosis were used to build diagnostic models with logistic regression (LR), naïve Bayes (nB), and multi-class perceptron (MLP). The diagnostic performances of the models were compared by ROC analysis using different train-test sampling approaches, including leave-one-out, 10-fold cross-validation, and varying percentage splits. METAVIR scoring was used for histological fibrosis staging of the liver. Results: 15 features showed a significant difference between the advanced and early liver fibrosis groups, p < 0.05. Among the individual features, first-order statics features led to the best classification with a sensitivity of 82.1−90.5% and a specificity of 87.1−89.8%. For the features combined, the diagnostic performances of nB and MLP were high, with the area under the ROC curve (AUC) approaching 0.95−0.96. LR also yielded high diagnostic performance (AUC = 0.91−0.92) but was lower than nB and MLP. The diagnostic variability between test-train trials, measured by the coefficient-of-variation (CV), was higher for LR (3−5%) than nB and MLP (1−2%). Conclusion: Quantitative ultrasound with machine learning differentiated early and advanced fibrosis. Ultrasound B-mode images contain a high level of information to enable accurate diagnosis with relatively straightforward machine learning methods like naïve Bayes and logistic regression. Implementing simple ML approaches with QUS features in clinical settings could reduce the user-dependent limitation of ultrasound in detecting early-stage liver fibrosis.

Can Sequential Images from the Same Object Be Used for Training Machine Learning Models? A Case Study for Detecting Liver Disease by Ultrasound Radiomics.

Machine learning for medical imaging not only requires sufficient amounts of data for training and testing but also that the data be independent. It is common to see highly interdependent data whenever there are inherent correlations between observations. This is especially to be expected for sequential imaging data taken from time series. In this study, we evaluate the use of statistical measures to test the independence of sequential ultrasound image data taken from the same case. A total of 1180 B-mode liver ultrasound images with 5903 regions of interests were analyzed. The ultrasound images were taken from two liver disease groups, fibrosis and steatosis, as well as normal cases. Computer-extracted texture features were then used to train a machine learning (ML) model for computer-aided diagnosis. The experiment resulted in high two-category diagnosis using logistic regression, with AUC of 0.928 and high performance of multicategory classification, using random forest ML, with AUC of 0.917. To evaluate the image region independence for machine learning, Jenson-Shannon (JS) divergence was used. JS distributions showed that images of normal liver were independent from each other, while the images from the two disease pathologies were not independent. To guarantee the generalizability of machine learning models, and to prevent data leakage, multiple frames of image data acquired of the same object should be tested for independence before machine learning. Such tests can be applied to real-world medical image problems to determine if images from the same subject can be used for training.

Machine learning improves early detection of liver fibrosis by quantitative ultrasound radiomics.

Progression of liver fibrosis to cirrhosis, a severe non-reversible process, is one of the most critical risk factors in developing hepatocellular carcinoma and liver failure. Detection of liver fibrosis at an early stage is therefore essential for better patient management. Ultrasound (US) imaging can provide a noninvasive alternative to biopsies. This study evaluates quantitative US texture features to improve early-stage versus advanced liver fibrosis detection. 157 B-mode US images of different liver lobes acquired from early and advanced fibrosis rat cases were used for analysis. 5-6 regions of interest were placed on each image. Twelve quantitative features that describe liver texture changes were extracted from the images, including first-order histogram, run length (RL), and gray level co-occurrence matrix (GLCM). The diagnostic performance of individual features was high with AUC ranging from 0.80 to 0.94. Logistic regression with leave-one-out cross-validation was used to evaluate the performance of the combined features. All features combined showed a slight improvement in performance with AUC = 0.95, sensitivity = 96.8%, and specificity = 93.7%. Quantitative US texture features characterize liver fibrosis changes with high accuracy and can differentiate early from advanced disease. Quantitative ultrasound, if validated in future clinical studies, can have a potential role in identifying fibrosis changes that are not easily detected by visual US image assessments.

Contrast-enhanced ultrasound for assessing blood flow modulation of hepatocellular carcinoma by hydralazine.

Modulating aberrant tumor microvasculature provides unique opportunities for enhancing ultrasound imaging of hepatocellular carcinoma (HCC). This study aims to use contrast-enhanced ultrasound to evaluate the potential of a potent vasodilator, hydralazine, to attenuate blood flow in HCC while enhancing it in the surrounding liver tissue. The "steel effect," where blood flow is diverted from the lesion to the surrounding tissue aims to enhance lesion-tissue contrast. Methods: HCC was induced in six rats by oral ingestion of diethylnitrosamine for 12 weeks. 10 tumors were studied to assess the enhancement in HCC tumors and surrounding tissue. Contrast-enhanced ultrasound images (CEUS) of each tumor were acquired before and after hydralazine injection. The enhancement of images was analyzed for the qualitative and quantitative assessment of HCC enhancement. Peak enhancement (PE) was calculated, representing the maximum signal intensity reached during the transit of the contrast bolus for both the tumor and the surrounding tissue. Intravenous administration of hydralazine significantly reduced CEUS signals in HCC tumors. The visual examination of images showed that the enhancement of tumors dramatically decreased after hydralazine injection. On the other hand, the surrounding tissue showed an increased enhancement. PE for the HCC changed from (71.8 ± 5) pre hydralazine to (28.7± 4.9), a 61.7% reduction after hydralazine injection, p=0.01. Future studies validating the technique in clinical settings for enhancing lesion-tissue contrast may allow physicians greater precision and accuracy in HCC surveillance for early detection of small tumors.

Hydralazine augmented ultrasound hyperthermia for the treatment of hepatocellular carcinoma.

This study investigates the use of hydralazine to enhance ultrasound hyperthermia for the treatment of hepatocellular carcinoma (HCC) by minimizing flow-mediated heat loss from the tumor. Murine HCC tumors were treated with a continuous mode ultrasound with or without an intravenous administration of hydralazine (5 mg/kg). Tumor blood flow and blood vessels were evaluated by contrast-enhanced ultrasound (CEUS) imaging and histology, respectively. Hydralazine markedly enhanced ultrasound hyperthermia through the disruption of tumor blood flow in HCC. Ultrasound treatment with hydralazine significantly reduced peak enhancement (PE), perfusion index (PI), and area under the curve (AUC) of the CEUS time-intensity curves by 91.9 ± 0.9%, 95.7 ± 0.7%, and 96.6 ± 0.5%, compared to 71.4 ± 1.9%, 84.7 ± 1.1%, and 85.6 ± 0.7% respectively without hydralazine. Tumor temperature measurements showed that the cumulative thermal dose delivered by ultrasound treatment with hydralazine (170.8 ± 11.8 min) was significantly higher than that without hydralazine (137.7 ± 10.7 min). Histological assessment of the ultrasound-treated tumors showed that hydralazine injection formed larger hemorrhagic pools and increased tumor vessel dilation consistent with CEUS observations illustrating the augmentation of hyperthermic effects by hydralazine. In conclusion, we demonstrated that ultrasound hyperthermia can be enhanced significantly by hydralazine in murine HCC tumors by modulating tumor blood flow. Future studies demonstrating the safety of the combined use of ultrasound and hydralazine would enable the clinical translation of the proposed technique.

Photoacoustic monitoring of oxygenation changes induced by therapeutic ultrasound in murine hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly vascular solid tumor. We have previously shown that ultrasound (US) therapy significantly reduces tumor vascularity. This study monitors US-induced changes in tumor oxygenation on murine HCC by photoacoustic imaging (PAI). Oxygen saturation and total hemoglobin were assessed by PAI before and after US treatments performed at different intensities of continuous wave (CW) bursts and pulsed wave (PW) bursts US. PAI revealed significant reduction both in HCC oxygen saturation and in total hemoglobin, proportional to the US intensity. Both CW bursts US (1.6 W/cm2) and the PW bursts US (0.8 W/cm2) significantly reduced HCC oxygen saturation and total hemoglobin which continued to diminish with time following the US treatment. The effects of US therapy were confirmed by power Doppler and histological examination of the hemorrhage in tumors. By each measure, the changes observed in US-treated HCC were more prevalent than those in sham-treated tumors and were statistically significant. In conclusion, the results show that US is an effective vascular-targeting therapy for HCC. The changes in oxygenation induced by the US treatment can be noninvasively monitored longitudinally by PAI without the use of exogenous image-enhancing agents. The combined use of PAI and the therapeutic US has potential for image-guided vascular therapy for HCC.

Subsequent Ultrasound Vascular Targeting Therapy of Hepatocellular Carcinoma Improves the Treatment Efficacy.

The response of hepatocellular carcinoma (HCC) to anti-vascular ultrasound therapy (AVUS) can be affected by the inherent differences in tumor vascular structure, and the functionality of tumor vessels at the time of treatment. In this study, we evaluate the hypothesis that repeated subsequent AVUS therapies are a possible approach to overcome these factors and improve the therapeutic efficacy of AVUS. HCC was induced in 30 Wistar rats by oral ingestion of diethylnitrosamine (DEN) for 12 weeks. A total of 24 rats received treatment with low intensity, 2.8 MHz ultrasound with an intravenous injection of microbubbles. Treated rats were divided into three groups: single therapy group (ST), 2-days subsequent therapy group (2DST), and 7-days subsequent therapy group (7DST). A sham control group did not receive ultrasound therapy. Tumor perfusion was measured by quantitative contrast-enhanced ultrasound (CEUS) nonlinear and power-Doppler imaging. Tumors were harvested for histologic evaluation of ultrasound-induced vascular changes. ANOVA was used to compare the percent change of perfusion parameters between the four treatment arms. HCC tumors treated with 2DST showed the largest reduction in tumor perfusion, with 75.3% reduction on average for all perfusion parameters. The ST group showed an average decrease in perfusion of 54.3%. The difference between the two groups was significant p < 0.001. The 7DST group showed a reduction in tumor perfusion of 45.3%, which was significant compared to the 2DST group (p < 0.001) but not different from the ST group (p = 0.2). The use of subsequent targeted AVUS therapies applied shortly (two days) after the first treatment enhanced the anti-vascular effect of ultrasound. This gain, however, was lost for a longer interval (1 week) between the therapies, possibly due to tumor necrosis and loss of tumor viability. These findings suggest that complex interplay between neovascularization and tumor viability plays a critical role in treatment and, therefore, must be actively monitored following treatment by CEUS for optimizing sequential treatment.

Photoacoustic Imaging for Assessing Tissue Oxygenation Changes in Rat Hepatic Fibrosis.

Chronic liver inflammation progressively evokes fibrosis and cirrhosis resulting in compromised liver function, and often leading to cancer. Early diagnosis and staging of fibrosis is crucial because the five-year survival rate of early-stage liver cancer is high. This study investigates the progression of hepatic fibrosis induced in rats following ingestion of diethylnitrosamine (DEN). Changes in oxygen saturation and hemoglobin concentration resulting from chronic inflammation were assayed longitudinally during DEN ingestion by photoacoustic imaging (PAI). Accompanying liver tissue changes were monitored simultaneously by B-mode sonographic imaging. Oxygen saturation and hemoglobin levels in the liver increased over 5 weeks and peaked at 10 weeks before decreasing at 13 weeks of DEN ingestion. The oxygenation changes were accompanied by an increase in hepatic echogenicity and coarseness in the ultrasound image. Histology at 13 weeks confirmed the development of severe fibrosis and cirrhosis. The observed increase in PA signal representing enhanced blood oxygenation is likely an inflammatory physiological response to the dietary DEN insult that increases blood flow by the development of neovasculature to supply oxygen to a fibrotic liver during the progression of hepatic fibrosis. Assessment of oxygenation by PAI may play an important role in the future assessment of hepatic fibrosis.

Dose-dependent effects of ultrasound therapy on hepatocellular carcinoma.

Non-invasive ischemic cancer therapy requires reduced blood flow whereas drug delivery and radiation therapy require increased tumor perfusion for a better response. In this study we investigate the hypothesis that different dose models of antivascular ultrasound therapy (AVUS) can have opposite effects on hepatocellular carcinoma (HCC) tumor blood flow. HCC was induced in 22 Wistar rats by ingestion of diethylnitrosamine (DEN) for 12 weeks. Rats received AVUS treatment at low and high doses. Low dose group received 1 watt/cm2 ultrasound for 1 min with 0.2 mL microbubbles injected IV. High dose group received 2 watts/cm2 for 2 min with 0.7 mL microbubbles IV. A sham group did not receive any treatment. Tumor perfusion was measured before and after AVUS with contrast-enhanced ultrasound. Quantitative perfusion measures: perfusion index (PI) and peak enhancement (PE) were obtained from each AVUS dose. After high-dose AVUS, PE and PI decreased by an average of 58.1 ± 4.9% and 49.1 ± 6.5 % respectively. Conversely, following low dose AVUS, PE and PI increased from baseline by an average of 47.8 ± 4.5% % and 20.3 ± 2.4 %, respectively. The high-dose AVUS therapy decreased tumoral perfusion, an effect that could be used for noninvasive ischemic therapy. Conversely, low-dose therapy increased tumor perfusion, which may improve drug delivery or radiation therapy. These opposite therapy effects can support multiple roles for AVUS in cancer therapy by tunable modulation of blood flow in tumors.

Microbubble-enhanced ultrasound for the antivascular treatment and monitoring of hepatocellular carcinoma.

Background and Objective: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and its current management relies heavily on locoregional therapy for curative therapy, bridge to transplant, and palliative therapy. Locoregional therapies include ablation and hepatic artery therapies such as embolization and radioembolization. In this study we evaluate the feasibility of using novel antivascular ultrasound (AVUS) as a noninvasive locoregional therapy to reduce perfusion in HCC lesions in a rat model and, monitor the effect with contrast-enhanced ultrasound imaging. Methods: HCC was induced in 36 Wistar rats by the ingestion of 0.01% diethylnitrosamine (DEN) for 12 weeks. Two therapy regimens of AVUS were evaluated. A primary regimen (n = 19) utilized 2-W/cm2, 3-MHz ultrasound (US) for 6 minutes insonation with 0.7 ml of microbubbles administered as an intravenous bolus. An alternate dose at half the primary intensity, sonication time, and contrast concentration was evaluated in 11 rats to assess the efficacy of a reduced dose. A control group (n = 6) received a sham therapy. Tumor perfusion was measured before and after AVUS with nonlinear contrast ultrasound (NLC) and power Doppler (PD). The quantitative perfusion measures included perfusion index (PI), peak enhancement (PE), time to peak (TTP), and perfusion area from NLC and PD scans. Total tumor area perfused during the scan was measured by a postprocessing algorithm called delta projection. Tumor histology was evaluated for signs of tissue injury and for vascular changes using CD31 immunohistochemistry. Results: DEN exposure induced autochthonous hepatocellular carcinoma lesions in all rats. Across all groups prior to therapy, there were no significant differences in the nonlinear contrast observations of peak enhancement and perfusion index. In the control group, there were no significant differences in any of the parameters after sham treatment. After the primary AVUS regimen, there were significant changes in all parameters (p ≤ 0.05) indicating substantial decreases in tumor perfusion. Peak enhancement in nonlinear contrast scans showed a 37.9% ± 10.1% decrease in tumor perfusion. Following reduced-dose AVUS, there were no significant changes in perfusion parameters, although there was a trend for the nonlinear contrast observations of peak enhancement and perfusion index to increase. Conclusion: This study translated low-intensity AVUS therapy into a realistic in vivo model of HCC and evaluated its effects on the tumor vasculature. The primary dose of AVUS tested resulted in significant vascular disruption and a corresponding reduction in tumor perfusion. A reduced dose of AVUS, on the other hand, was ineffective at disrupting perfusion but demonstrated the potential for enhancing tumor blood flow. Theranostic ultrasound, where acoustic energy and microbubbles are used to monitor the tumor neovasculature as well as disrupt the vasculature and treat lesions, could serve as a potent tool for delivering noninvasive, locoregional therapy for hepatocellular carcinoma.

Photoacoustic and Ultrasound Dual-Mode Imaging via Functionalization of Recombinant Protein-Stabilized Microbubbles with Methylene Blue.

Contrast-enhanced photoacoustics and ultrasonics are complementary methods of bioimaging. In this study, a flow-focusing junction microfluidic device is used for the generation of uniform microbubbles (<5 µm) for simultaneous enhancement of photoacoustic and ultrasound imaging. Microbubbles stabilized with a mixture of a recombinant protein and a synthetic amphiphilic block copolymer are functionalized with an FDA-approved photoacoustic dye, methylene blue (MetB). These microbubbles are uniform in size and stable. We show that the ultrasound and photoacoustic signals can be independently controlled by changing the concentration of MetB during microbubble preparation and the concentration of MetB-functionalized microbubbles in the probe suspension. We also perform animal tests to demonstrate the enhancement of ultrasound and acoustic signals upon injection of MetB-functionalized microbubbles in mice. The increase in the sonographic and photoacoustic signals is visibly obvious in the images. Taken together, MetB-functionalized microbubbles represent promising dual-mode ultrasound and photoacoustic imaging contrast agents for theranostic applications.

Bioinformatic identification of novel protein phosphatases in the dog genome.

Protein kinases and protein phosphatases constitute about 2-4% of the genes in a typical eukaryotic genome. Protein phosphatases are important players in many cellular processes such as proliferation, differentiation, cell adhesion, and motility. In this study, we identified, classified, and analyzed protein phosphatase complement of the dog genome. In this article, we report the identification of at least 178 putative protein phosphatases in dog which include 51 PSTPs, 112 PTPs, and 15 Asp-based protein phosphatases. Interestingly, we found at least five novel protein phosphatases in dog, namely DUSP5L, DUSP18L, MTMR9L, MTMR12L, and PPP6CL which are not present in human, mouse, rat, and cow. In addition, we found PTP4A1-rt, a retro-transposed copy of the PTP4A1 gene, in chromosome 27. Furthermore, we modeled three-dimensional structures of the catalytic domains of these putative protein phosphatases and aligned them to see the structural similarities between them. We docked PPP2CA with okadaic acid and calculated the value of affinity energy as -8.8 kcal/mol. Our nucleotide substitution rate study revealed that apparently none of the phosphatase family is under significantly higher evolutionary pressure.

Mutation of the hydrophobic motif in a phosphorylation-deficient mutant renders protein kinase C delta more apoptotically active.

Protein kinase C delta (PKCdelta) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCdelta is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCdelta. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCdelta activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCdelta (PKCdelta-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCdelta apoptotically more active. Furthermore, we found that the mutant PKCdelta-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart.