Gasdermin D-mediated metabolic crosstalk promotes tissue repair.

The establishment of an early pro-regenerative niche is crucial for tissue regeneration1,2. Gasdermin D (GSDMD)-dependent pyroptosis accounts for the release of inflammatory cytokines upon various insults3-5. However, little is known about its role in tissue regeneration followed by homeostatic maintenance. Here, we show that macrophage GSDMD deficiency delayed tissue recovery, with little impact on the local inflammatory milieu or the lytic pyroptosis process. Metabolite secretome profiling of hyperactivated macrophages unveiled the non-canonical metabolite-secreting function of GSDMD. And we further identified 11,12-epoxyeicosatrienoic acid (11,12-EET) as a bioactive pro-healing oxylipin, secreted from hyperactive macrophages in a GSDMD-dependent manner. Indeed, accumulation of 11,12-EET by direct supplementation or deletion of its hydrolytic enzyme Ephx2 accelerated muscle regeneration. We further demonstrated that the Ephx2 level accumulated within aged muscle. And consecutive 11,12-EET treatment rejuvenated aged muscle. Mechanistically, 11,12-EET amplifies FGF-FGFR signaling by modulating FGF liquid-liquid phase separation, hence boosting the activation and proliferation of muscle stem cells (MuSCs). These data depict a GSDMD-guided metabolite crosstalk between macrophages and MuSCs that governs the repair process, which offers new therapeutic insights for the regeneration of injured or aged tissues.

Prognostic significance of dynamic changes in liver stiffness measurement in patients with chronic hepatitis B and compensated advanced chronic liver disease.

BACKGROUND AND AIM: Liver stiffness measurements (LSMs) are promising for monitoring disease progression or regression. We assessed the prognostic significance of dynamic changes in LSM over time on liver-related events (LREs) and death in patients with chronic hepatitis B (CHB) and compensated advanced chronic liver disease (cACLD). METHODS: This retrospective study included 1272 patients with CHB and cACLD who underwent at least two measurements, including LSM and fibrosis score based on four factors (FIB-4). ΔLSM was defined as [(follow-up LSM - baseline LSM)/baseline LSM × 100]. We recorded LREs and all-cause mortality during a median follow-up time of 46 months. Hazard ratios (HRs) and confidence intervals (CIs) for outcomes were calculated using Cox regression. RESULTS: Baseline FIB-4, baseline LSM, ΔFIB-4, ΔLSM, and ΔLSM/year were independently and simultaneously associated with LREs (adjusted HR, 1.04, 95% CI, 1.00-1.07; 1.02, 95% CI, 1.01-1.03; 1.06, 95% CI, 1.03-1.09; 1.96, 95% CI, 1.63-2.35, 1.02, 95% CI, 1.01-1.04, respectively). The baseline LSM combined with the ΔLSM achieved the highest Harrell's C (0.751), integrated AUC (0.776), and time-dependent AUC (0.737) for LREs. Using baseline LSM and ΔLSM, we proposed a risk stratification method to improve clinical applications. The risk proposed stratification based on LSM performed well in terms of prognosis: low risk (n = 390; reference), intermediate risk (n = 446; HR = 3.38), high risk (n = 272; HR = 5.64), and extremely high risk (n = 164; HR = 11.11). CONCLUSIONS: Baseline and repeated noninvasive tests measurement allow risk stratification of patients with CHB and cACLD. Combining baseline and dynamic changes in the LSM improves prognostic prediction.

The steatosis-associated fibrosis estimator (SAFE) outperformed the FIB-4 score in screening the population for liver disease.

INTRODUCTION AND OBJECTIVES: Assessing fibrosis risk noninvasively is essential. The steatosis-associated fibrosis estimator (SAFE) score shows promise but needs validation. PATIENTS AND METHODS: This was a three-part study. In part 1, we compared the SAFE score with the Fibrosis-4 (FIB-4) and NAFLD fibrosis score (NFS) in the National Health and Nutrition Examination Survey (NHANES) cohort (2017-2020), using transient elastography (TE) as screening reference. In part 2, we examined patients who underwent liver biopsies at an Asian center between 2018 and 2020 to assess these models in various liver diseases. In part 3, the SAFE score was applied to adults in the NHANES cohort (1999-2016) to assess the correlation with mortality. RESULTS: In part 1, we studied 6,677 patients, comprising 595 screening positive (TE ≥8 kPa). SAFE (cutoff 100) displayed a lower proportion of false positives (10.4 %) than FIB-4 (cutoff 1.3) and NFS (cutoff -1.455) (22.1 % and 43.6 %) while retaining a low proportion of false negatives (5.5 %). In part 2, SAFE outperformed FIB-4 (P = 0.04) and NFS (P = 0.04) in staging significant fibrosis (≥S2) in NAFLD and had similar accuracies in other etiologies. In part 3, the FIB-4, NFS, and SAFE score were associated with all-cause mortality in the general population, with c-statistics of 0.738, 0.736, and 0.759, respectively. CONCLUSIONS: The SAFE score reduced futile referrals more effectively than FIB-4 without raising the missed TE ≥ 8 kPa rate. It correlated with all-cause mortality in the general population and excelled in staging significant fibrosis in NAFLD.

p-d Orbital Hybridization in Ag-based Electrocatalysts for Enhanced Nitrate-to-Ammonia Conversion.

Considering the substantial role of ammonia, developing highly efficient electrocatalysts for nitrate-to-ammonia conversion has attracted increasing interest. Herein, we proposed a feasible strategy of p-d orbital hybridization via doping p-block metals in an Ag host, which drastically promotes the performance of nitrate adsorption and disassociation. Typically, a Sn-doped Ag catalyst (SnAg) delivers a maximum Faradaic efficiency (FE) of 95.5±1.85 % for NH3 at -0.4 V vs. RHE and reaches the highest NH3 yield rate to 482.3±14.1 mg h-1 mgcat. -1. In a flow cell, the SnAg catalyst achieves a FE of 90.2 % at an ampere-level current density of 1.1 A cm-2 with an NH3 yield of 78.6 mg h-1 cm-2, during which NH3 can be further extracted to prepare struvite as high-quality fertilizer. A mechanistic study reveals that a strong p-d orbital hybridization effect in SnAg is beneficial for nitrite deoxygenation, a rate-determining step for NH3 synthesis, which as a general principle, can be further extended to Bi- and In-doped Ag catalysts. Moreover, when integrated into a Zn-nitrate battery, such a SnAg cathode contributes to a superior energy density of 639 Wh L-1, high power density of 18.1 mW cm-2, and continuous NH3 production.

Emerging Electrocatalysts in Urea Production.

Urea synthesis from abundant CO2 and N-feedstocks via renewable electricity has attracted increasing interests, offering a promising alternative to the industrial-applied Haber-Meiser process. However, the studies toward electrochemical urea production remain scarce and appeal for more research. Herein, in this perspective, an up-to-date overview on the urea electrosynthesis is highlighted and summarized. Firstly, the reaction pathways of urea formation through various feedstocks are comprehensively discussed. Then, we focus on the strategies of materials design to improve C-N coupling efficiency by identifying the descriptor and understanding the reaction mechanism. Finally, the current challenges and disadvantages in this field are reviewed and some future development directions of electrocatalytic urea synthesis are also prospected. This Minireview aims to promote future investigations of the electrochemical urea synthesis.

A partial convolution generative adversarial network for lesion synthesis and enhanced liver tumor segmentation.

Lesion segmentation is critical for clinicians to accurately stage the disease and determine treatment strategy. Deep learning based automatic segmentation can improve both the segmentation efficiency and accuracy. However, training a robust deep learning segmentation model requires sufficient training examples with sufficient diversity in lesion location and lesion size. This study is to develop a deep learning framework for generation of synthetic lesions with various locations and sizes that can be included in the training dataset to enhance the lesion segmentation performance. The lesion synthesis network is a modified generative adversarial network (GAN). Specifically, we innovated a partial convolution strategy to construct a U-Net-like generator. The discriminator is designed using Wasserstein GAN with gradient penalty and spectral normalization. A mask generation method based on principal component analysis (PCA) was developed to model various lesion shapes. The generated masks are then converted into liver lesions through a lesion synthesis network. The lesion synthesis framework was evaluated for lesion textures, and the synthetic lesions were used to train a lesion segmentation network to further validate the effectiveness of the lesion synthesis framework. All the networks are trained and tested on the LITS public dataset. Our experiments demonstrate that the synthetic lesions generated by our approach have very similar distributions for the two parameters, GLCM-energy and GLCM-correlation. Including the synthetic lesions in the segmentation network improved the segmentation dice performance from 67.3% to 71.4%. Meanwhile, the precision and sensitivity for lesion segmentation were improved from 74.6% to 76.0% and 66.1% to 70.9%, respectively. The proposed lesion synthesis approach outperforms the other two existing approaches. Including the synthetic lesion data into the training dataset significantly improves the segmentation performance.

Dosimetry impact of distinct gating strategies in cine MR image-guided breath-hold pancreatic cancer radiotherapy.

PURPOSE: To investigate the dosimetry effects of different gating strategies in cine magnetic resonance imaging (MRI)-guided breath-hold pancreatic cancer radiotherapy. METHODS: Two cine MRI-based gating strategies were investigated： a tumor contour-based gating strategy at a gating threshold of 0-5% and a tumor displacement-based gating strategy at a gating threshold of 3-5 mm. The cine MRI videos were obtained from 17 pancreatic cancer patients who received MRI-guided radiation therapy. We calculated the tumor displacement in each cine MR frame that satisfied the gating threshold and obtained the proportion of frames with different displacements. We generated IMRT and VMAT plans using a 33 Gy prescription, and motion plans were generated by adding up all isocenter-shift plans corresponding to different tumor displacements. The dose parameters of GTV, PTV, and organs at risk (OAR) were compared between the original and motion plans. RESULTS: In both gating strategies, the difference was significant in PTV coverage but not in GTV coverage between the original and motion plans. OAR dose parameters deteriorate with increasing gating threshold. The beam duty cycle increased from 19.5±14.3% (median 18.0%) to 60.8±15.6% (61.1%) for gating thresholds from 0% to 5% in tumor contour-based gating and from 51.7±11.5% (49.7%) to 67.3±12.4% (67.1%) for gating thresholds from 3 to 5 mm in tumor displacement-based gating. CONCLUSION: In tumor contour-based gating strategy, the dose delivery accuracy deteriorates while the dose delivery efficiency improves with increasing gating thresholds. To ensure treatment efficiency, the gating threshold might be no less than 3%. A threshold up to 5% may be acceptable in terms of the GTV coverage. The displacement-based gating strategy may serve as a potential alternative to the tumor contour based gating strategy, in which the gating threshold of approximately 4 mm might be a good choice for reasonably balancing the dose delivery accuracy and efficiency.

Carbon-Anchored Molybdenum Oxide Nanoclusters as Efficient Catalysts for the Electrosynthesis of Ammonia and Urea.

The electrochemical NO3 - reduction and its coupling with CO2 can provide novel and clean routes to synthesize NH3 and urea, respectively. However, their practical application is still impeded by the lack of efficient catalysts with desirable Faradaic efficiency (FE) and yield rate. Herein, we report the synthesis of molybdenum oxide nanoclusters anchored on carbon black (MoOx /C) as electrocatalyst. It affords an outstanding FE of 98.14 % and NH3 yield rate of 91.63 mg h-1 mgcat. -1 in NO3 - reduction. Besides, the highest FE of 27.7 % with a maximum urea yield rate of 1431.5 µg h-1 mgcat. -1 toward urea is also achieved. The formation of electron-rich MoOx nanoclusters with highly unsaturated metal sites in the MoOx /C heterostructure is beneficial for enhanced catalytic performance. Studies on the mechanism reveal that the stabilization of *NO and *CO2 NOOH intermediates are critical for the NH3 and urea synthesis, respectively.

Quantitative x-ray fluorescence imaging system for non-destructive 3D tumor histology.

An in-house dual-modality x-ray fluorescence tomography (XFT) and x-ray computed tomography (XCT) system was developed to quantify iodine contrast distribution through the whole tumor volume ex vivo. The quantitative XFT was calibrated with water phantoms containing iodine solutions of various concentrations (0.0175-1.4 wt.%). The vasculature distribution was reflected by the iodine perfusion, which was validated with histology. This technique may open a new, to the best of our knowledge, route to the non-destructive three-dimensional-imaging-based histological analysis of tumor samples.

Automatic treatment planning for cervical cancer radiation therapy using direct three-dimensional patient anatomy match.

PURPOSE: Current knowledge-based planning methods for radiation therapy mainly use low-dimensional features extracted from contoured structures to identify geometrically similar patients. Here, we propose a knowledge-based treatment planning method where the anatomical similarity is quantified by the rigid registration of the three-dimensional (3D) planning target volume (PTV) and organs at risks (OARs) between an incoming patient and database patients. METHODS: A database that contains PTV and OARs contours from 81 cervical cancer radiation therapy patients was established. To identify the anatomically similar patients, the PTV of the new patient was registered to each PTV in the database and the Dice similarity coefficients were calculated for the PTV, rectum, and bladder between the new patient and database patients. Then the top 20 patients in the PTV match and top 3 patients in the subsequent bladder or rectum match were selected. The best dose-volume histogram parameters from the top three patients were applied as the dose constraints to the automatic plan optimization. A fast Fourier transform algorithm was developed to accelerate the 3D PTV registration process run through the database. The entire treatment planning process was automated using in-house customized Pinnacle scripts. The automatic plans were generated for 20 patients using leave-one-out scheme and were evaluated against the corresponding clinical plans. RESULTS: The automatic plans significantly reduced rectum and bladder V 50 Gy ${V_{50\,\,{\rm{Gy}}}}$ by 11.79% ± 5.2% (p < 0.01) and 2.85% ± 3.16% (p < 0.01), respectively. The dose parameters achieved for the PTV and other OARs were comparable to those in the clinical plans. The entire planning process, including both dose prediction and inverse optimization, costs about 6 min. CONCLUSIONS: The direct 3D contour match method utilizes the full spatial information of the PTV and OARs of interest and provides an intuitive measurement for patient plan anatomy similarity. The proposed automatic planning method can generate plans with better quality and higher efficiency.

Development of an unsupervised cycle contrastive unpaired translation network for MRI-to-CT synthesis.

PURPOSE: The purpose of this work is to develop and evaluate a novel cycle-contrastive unpaired translation network (cycleCUT) for synthetic computed tomography (sCT) generation from T1-weighted magnetic resonance images (MRI). METHODS: The cycleCUT proposed in this work integrated the contrastive learning module from contrastive unpaired translation network (CUT) into the cycle-consistent generative adversarial network (cycleGAN) framework to effectively achieve unsupervised CT synthesis from MRI. The diagnostic MRI and radiotherapy planning CT images of 24 brain cancer patients were obtained and reshuffled to train the network. For comparison, the traditional cycleGAN and CUT were also implemented. The sCT images were then imported into a treatment planning system to verify their feasibility for radiotherapy planning. The mean absolute error (MAE), peak signal-to-noise ratio (PSNR), and structural similarity index (SSIM) between the sCT and the corresponding real CT images were calculated. Gamma analysis between sCT- and CT-based dose distributions was also conducted. RESULTS: Quantitative evaluation of an independent test set of six patients showed that the average MAE was 69.62 ± 5.68 Hounsfield Units (HU) for the proposed cycleCUT, significantly (p-value < 0.05) lower than that for cycleGAN (77.02 ± 6.00 HU) and CUT (78.05 ± 8.29). The average PSNR was 28.73 ± 0.46 decibels (dB) for cycleCUT, significantly larger than that for cycleGAN (27.96 ± 0.49 dB) and CUT (27.95 ± 0.69 dB). The average SSIM for cycleCUT (0.918 ± 0.012) was also significantly higher than that for cycleGAN (0.906 ± 0.012) and CUT (0.903 ± 0.015). Regarding gamma analysis, cycleCUT achieved the highest passing rate (97.95 ± 1.24% at the 2%/2 mm criteria and 10% dose threshold) but was not significantly different from the others. CONCLUSION: The proposed cycleCUT could be effectively trained using unaligned image data, and could generate better sCT images than cycleGAN and CUT in terms of HU number accuracy and fine structural details.

Can you voluntarily forget what you are planning to forget? Behavioral evidence for the underlying truth of the cost-benefit principle.

Directed forgetting (DF) or intentional forgetting can impair the recall of items cued to be forgotten (so-called cost) but can also improve the recall of items cued to be remembered (so-called benefit), following the cost-benefit principle. However, whether the forget cue is the cause of intentional forgetting is doubtful. Several studies suggest that intentional forgetting is extremely hard by introducing a noncue condition, but it is not enough to explain how the cost-benefit principle works without forgetting. Here, two series of experiments are designed to test the voluntary control ability of attentional resource allocation from memory benefits to costs. In the Experiment 1 series, we changed the position and content of cues, replicating the DF effect and the analogous effect without a forget cue. The results showed that precueing can mildly upregulate memory attentional resources to improve performance while incurring memory costs for other items. Since forced remembering can also induce memory costs and benefits, the cost-benefit principle may not be attributed to DF. In the Experiment 2 series, we further demonstrated that memory costs and benefits exist when implicit cues are employed. Overall, when the results of all memory costs in the above experiments are compared with a noncue condition as the baseline in Experiment 2c, there exist no significant differences. The current results indicate that we can voluntarily upregulate our memory performance for certain items but cannot voluntarily downregulate memory encoding, generalizing the selective attentional resource control account to the cost-benefit principle beyond the directed-forgetting effect.

ß-Arrestin1 is involved in hepatocellular carcinoma metastasis via extracellular signal-regulated kinase-mediated epithelial-mesenchymal transition.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a malignant disease worldwide. It is implicated in high cancer-related mortality rates in humans. ß-Arrestin1 (ARRB1) has been demonstrated to be related to the development of several cancers, while the relationship between ARRB1 and metastasis in HCC is unknown. METHODS: A tissue microarray of 68 tissues from HCC patients with or without metastasis was collected. Wild-type and ARRB1 knockout mice were used to examine the role of ARRB1 in metastasis in vivo. The level of ARRB1 in HCC tissues, mouse liver tissues, and cell lines was determined by quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. Migration, invasion, and motility capacities of HCC cells were determined by transwell assay and wound healing assay. Vein injection of nude mice model was used to reveal the metastatic abilities of HCC cell lines. For the mechanism study, we investigated the effects of ARRB1 on the phosphorylation of ERK1/2 and the expression of epithelial-mesenchymal transition (EMT) markers in HCC. RESULTS: We reveal that ARRB1 accelerates metastasis in HCC and that ARRB1 deficiency inhibits hepatocarcinogenesis and reverses EMT in mice. ARRB1 regulates HCC cell migration and invasion and suppresses HCC metastasis in vivo. Furthermore, we show that ARRB1 promotes EMT through the phosphorylation of ERK1/2. CONCLUSIONS: Our data suggest that ARRB1 promotes HCC invasion and metastasis through p-ERK1/2-mediated EMT and that suppression of ARRB1 or p-ERK1/2 may offer potential therapeutic targets for HCC therapy.

Targeting mitochondria-associated membranes as a potential therapy against endothelial injury induced by hypoxia.

Mitochondrial dysfunction plays a principal role in hypoxia-induced endothelial injury, which is involved in hypoxic pulmonary hypertension and ischemic cardiovascular diseases. Recent studies have identified mitochondria-associated membranes (MAMs) that modulate mitochondrial function under a variety of pathophysiological conditions such as high-fat diet-mediated insulin resistance, hypoxia reoxygenation-induced myocardial death, and hypoxia-evoked vascular smooth muscle cell proliferation. However, the role of MAMs in hypoxia-induced endothelial injury remains unclear. To explore this further, human umbilical vein endothelial cells and human pulmonary artery endothelial cells were exposed to hypoxia (1% O2 ) for 24 hours. An increase in MAM formation was uncovered by immunoblotting and immunofluorescence. Then, we performed small interfering RNA transfection targeted to MAM constitutive proteins and explored the biological effects. Knockdown of MAM constitutive proteins attenuated hypoxia-induced elevation of mitochondrial Ca2+ and repressed mitochondrial impairment, leading to an increase in mitochondrial membrane potential and ATP production and a decline in reactive oxygen species. Then, we found that MAM disruption mitigated cell apoptosis and promoted cell survival. Next, other protective effects, such as those pertaining to the repression of inflammatory response and the promotion of NO synthesis, were investigated. With the disruption of MAMs under hypoxia, inflammatory molecule expression was repressed, and the eNOS-NO pathway was enhanced. This study demonstrates that the disruption of MAMs might be of therapeutic value for treating endothelial injury under hypoxia, suggesting a novel strategy for preventing hypoxic pulmonary hypertension and ischemic injuries.

Increased expression of GATA zinc finger domain containing 1 through gene amplification promotes liver cancer by directly inducing phosphatase of regenerating liver 3.

We identified that GATA zinc finger domain containing 1 (GATAD1), a transcriptional factor, was significantly up-regulated in hepatocellular carcinoma (HCC) through gene amplification. We demonstrated the critical role, molecular mechanisms, and clinical implications of GATAD1 as a novel oncogenic factor in HCC. We found that GATAD1 protein was expressed in 76.6% of primary HCCs (85/111) but silenced in normal liver tissues. Gene amplification of GATAD1 was positively correlated with its overexpression in primary HCCs (R = 0.629, P < 0.0001). GATAD1 significantly increased cell proliferation, G1 -S cell cycle transition, and migration/invasion but suppressed apoptosis in liver cell lines and promoted tumor growth and lung metastasis in both xenograft and orthotopic mouse models. Mechanistically, GATAD1 induced the transcriptional expression of phosphatase of regenerating liver 3 (PRL3) by binding to its promoter identified by RNA sequencing and chromatin immunoprecipitation-PCR analyses. PRL3 played an oncogenic role in HCC. Knockdown of PRL3 blunted the tumorigenic effect of GATAD1. In addition, GATAD1 activated Akt signaling, evidenced by increased phosphorylation levels of total Akt, Akt1, Akt2, and Akt target glycogen synthase kinase 3ß, while knockdown of PRL3 abolished this effect of GATAD1. We further unveiled that PRL3 activated Akt signaling by dephosphorylating phosphatase and tensin homolog at tyrosine residue, thus reducing phosphatase and tensin homolog protein. The PRL3 inhibitor 5-[[5-bromo-2-[(2-bromophenyl)methoxy]phenyl]methylene]-2-thioxo-4-thiazolidinone significantly suppressed HCC growth by inhibiting Akt activation. Moreover, high GATAD1 nuclear protein expression was associated with poor survival of HCC patients as an independent prognostic factor. CONCLUSION: GATAD1 plays a pivotal oncogenic role in HCC by directly inducing PRL3 transcription to activate the Akt signaling pathway. GATAD1 may serve as an independent poor prognostic factor for HCC patients. (Hepatology 2018;67:2302-2319).

Nogo-B Receptor Directs Mitochondria-Associated Membranes to Regulate Vascular Smooth Muscle Cell Proliferation.

Mitochondria-associated membranes (MAM) are a well-recognized contact link between the mitochondria and endoplasmic reticulum that affects mitochondrial biology and vascular smooth muscle cells (VSMCs) proliferation via the regulation of mitochondrial Ca2+(Ca2+m) influx. Nogo-B receptor (NgBR) plays a vital role in proliferation, epithelial-mesenchymal transition, and chemoresistance of some tumors. Recent studies have revealed that downregulation of NgBR, which stimulates the proliferation of VSMCs, but the underlying mechanism remains unclear. Here, we investigated the role of NgBR in MAM and VSMC proliferation. We analyzed the expression of NgBR in pulmonary arteries using a rat model of hypoxic pulmonary hypertension (HPH), in which rats were subjected to normoxic recovery after hypoxia. VSMCs exposed to hypoxia and renormoxia were used to assess the alterations in NgBR expression in vitro. The effect of NgBR downregulation and overexpression on VSMC proliferation was explored. The results revealed that NgBR expression was negatively related with VSMCs proliferation. Then, MAM formation and the phosphorylation of inositol 1,4,5-trisphosphate receptor type 3 (IP3R3) was detected. We found that knockdown of NgBR resulted in MAM disruption and augmented the phosphorylation of IP3R3 through pAkt, accompanied by mitochondrial dysfunction including decreased Ca2+m, respiration and mitochondrial superoxide, increased mitochondrial membrane potential and HIF-1α nuclear localization, which were determined by confocal microscopy and Seahorse XF-96 analyzer. By contrast, NgBR overexpression attenuated IP3R3 phosphorylation and HIF-1α nuclear localization under hypoxia. These results reveal that dysregulation of NgBR promotes VSMC proliferation via MAM disruption and increased IP3R3 phosphorylation, which contribute to the decrease of Ca2+m and mitochondrial impairment.

Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes.

Autophagy and apoptosis are interlocked in an extensive crosstalk. Our previous study demonstrated that hypotonic hypoxia-induced marked apoptosis of a spermatocyte-derived cell line (GC-2). However, whether hypoxia-induced apoptosis is mediated by inhibition of autophagy under hypoxic conditions remains unclear. In this study, GC-2 cells were cultured in 1% O2 and harvested at different time points. Autophagy was determined by acridine orange staining, cyto-ID staining, mCherry-GFP-LC3B adenovirus transfection and Western blotting for various autophagy markers. Apoptosis was detected by TUNEL staining, flow cytometry, JC-1 staining and Western blotting of apoptosis-related proteins. We found that hypoxia-induced apoptosis of GC-2 cells through mitochondrial and death receptor pathways and inhibited autophagic flux in GC-2 cells in a time-dependent manner. However, while marked autolysosome formation was observed in GC-2 cells before 24-h culture in hypoxic conditions, apparent apoptosis was observed only after 24-h culture in hypoxic conditions. Caspase-8 siRNA treatment induced cell survival, accompanied by induction of the mature autophagosome, acidic vesicular organelle formation and autophagic flux. Furthermore, Beclin-1 overexpression markedly attenuated the impairment of spermatogenesis in mice by inhibiting apoptosis of spermatocytes. The results of this study demonstrate that hypoxia inhibits autophagy, which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner, suggesting that combined application of apoptosis inhibition and autophagy activation might be a therapeutic strategy for treating hypoxia-induced male infertility.

Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.

The molecular mechanisms responsible for the development of proteinuria and glomerulosclerosis in radiation nephropathy remain largely unknown. Podocytes are increasingly recognized as key players in the pathogenesis of proteinuria in primary and secondary glomerular disorders. The lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) is a key determinant of podocyte injury and a known off target of the anti-CD20 antibody rituximab (RTX). The current study investigates the role of sphingolipids in radiation-induced podocytopathy. After a single dose of radiation (8 Gy), several ceramide species were significantly elevated. In particular, C16:00, C24:00, and C24:1 ceramides were the most abundant ceramide species detected. These changes were paralleled by a time-dependent drop in SMPDL3b protein, sphingosine, and sphingosine-1-phosphate levels. Interestingly, SMPDL3b-overexpressing podocytes had higher basal levels of sphingosine-1-phosphate and maintained basal ceramide levels after irradiation. Morphologically, irradiated podocytes demonstrated loss of filopodia and remodeling of cortical actin. Furthermore, the actin binding protein ezrin relocated from the plasma membrane to the cytosol as early as 2 h after radiation. In contrast, SMPDL3b overexpressing podocytes were protected from radiation-induced cytoskeletal remodeling. Treatment with RTX before radiation exposure partially protected podocytes from SMPDL3b loss, cytoskeletal remodeling, and caspase 3 cleavage. Our results demonstrate that radiation injury induces early cytoskeletal remodeling, down-regulation of SMPDL3b, and elevation of cellular ceramide levels. Overexpression of SMPDL3b and pretreatment with RTX confer a radioprotective effect in cultured podocytes. These findings indicate a potential role for SMPDL3b and RTX in radiation-induced podocytopathy.-Ahmad, A., Mitrofanova, A., Bielawski, J., Yang, Y., Marples, B., Fornoni, A., Zeidan, Y. H. Sphingomyelinase-like phosphodiesterase 3b mediates radiation-induced damage of renal podocytes.

Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth.

Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia-induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI-treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia-induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.

PDGF-BB and bFGF ameliorate radiation-induced intestinal progenitor/stem cell apoptosis via Akt/p53 signaling in mice.

Radiation-induced gastrointestinal (GI) syndrome currently has no effective prophylactic or therapeutic treatment. Previous studies and our data have demonstrated the important role of p53 in acute radiation-induced GI syndrome in mice. Many cytokines, such as tumor necrosis factor-α and fibroblast growth factor (bFGF), have been found to protect against radiation-induced intestinal injury, although the underlying mechanisms remain to be identified. Here, we report blockage of p53 through a protein kinase B (Akt) pathway in intestinal progenitor/stem cells or crypt cells as a novel molecular mechanism of growth factor-mediated intestinal radioprotection. Treatment with platelet-derived growth factor (PDGF-BB) or bFGF activated Akt phosphorylation in the intestinal crypt, lessened intestinal crypt p53 expression, decreased radiation-induced apoptosis in mouse intestinal progenitor/stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive cells by an average of 50%, and increased the survival rate of mice with abdominal radiation by 3 days in average. Conversely, the Akt inhibitor perifosine obstructed growth factor-simulated Akt phosphorylation while promoting radiation-induced p53 expression in intestinal crypts. Importantly, reduced Akt phosphorylation and elevated p53 expression due to the Akt inhibitor perifosine impaired intestinal progenitor/stem cells radioprotection provided by PDGF-BB and bFGF. Consistently, PDGF-BB and bFGF both upregulated Akt activation, suppressed radiation-induced p53 expression, and abrogated radiation-induced apoptosis in IEC-6 cells, although p53 overexpression in IEC-6 cells partially counteracted the radioprotection of PDGF-BB and bFGF. Our data suggest that intestinal crypt radioprotection by PDGF-BB and bFGF is dependent on regulation of Akt/p53 signaling.