Fluctuations in p53 Signaling Allow Escape from Cell-Cycle Arrest.

Biological signals need to be robust and filter small fluctuations yet maintain sensitivity to signals across a wide range of magnitudes. Here, we studied how fluctuations in DNA damage signaling relate to maintenance of long-term cell-cycle arrest. Using live-cell imaging, we quantified division profiles of individual human cells in the course of 1 week after irradiation. We found a subset of cells that initially establish cell-cycle arrest and then sporadically escape and divide. Using fluorescent reporters and mathematical modeling, we determined that fluctuations in the oscillatory pattern of the tumor suppressor p53 trigger a sharp switch between p21 and CDK2, leading to escape from arrest. Transient perturbation of p53 stability mimicked the noise in individual cells and was sufficient to trigger escape from arrest. Our results show that the self-reinforcing circuitry that mediates cell-cycle transitions can translate small fluctuations in p53 signaling into large phenotypic changes.

Single-cell RNA sequencing deciphers the mechanism of sepsis-induced liver injury and the therapeutic effects of artesunate.

Liver, as an immune and detoxification organ, represents an important line of defense against bacteria and infection and a vulnerable organ that is easily injured during sepsis. Artesunate (ART) is an anti-malaria agent, that also exhibits broad pharmacological activities including anti-inflammatory, immune-regulation and liver protection. In this study, we investigated the cellular responses in liver to sepsis infection and ART hepatic-protective mechanisms against sepsis. Cecal ligation and puncture (CLP)-induced sepsis model was established in mice. The mice were administered ART (10 mg/kg, i.p.) at 4 h, and sacrificed at 12 h after the surgery. Liver samples were collected for preparing single-cell RNA transcriptome sequencing (scRNA-seq). The scRNA-seq analysis revealed that sepsis-induced a dramatic reduction of hepatic endothelial cells, especially the subtypes characterized with proliferation and differentiation. Macrophages were recruited during sepsis and released inflammatory cytokines (Tnf, Il1b, Il6), chemokines (Ccl6, Cd14), and transcription factor (Nfkb1), resulting in liver inflammatory responses. Massive apoptosis of lymphocytes and abnormal recruitment of neutrophils caused immune dysfunction. ART treatment significantly improved the survival of CLP mice within 96 h, and partially relieved or reversed the above-mentioned pathological features, mitigating the impact of sepsis on liver injury, inflammation, and dysfunction. This study provides comprehensive fundamental proof for the liver protective efficacy of ART against sepsis infection, which would potentially contribute to its clinical translation for sepsis therapy. Single cell transcriptome reveals the changes of various hepatocyte subtypes of CLP-induced liver injury and the potential pharmacological effects of artesunate on sepsis.

Translation and validation of a Chinese version of the pancreatitis quality of life instrument (PANQOLI) in patients with chronic pancreatitis.

PURPOSE: Chronic pancreatitis (CP) is a chronic fibroinflammatory pancreatic disease that severely impacts patients' quality of life (QoL). The Pancreatitis Quality of Life Instrument (PANQOLI) is an 18-item measure specifically designed to assess QoL amongst patients with CP. This study aimed to develop a Chinese version of PANQOLI and assess its reliability and validity in the Chinese CP cohort. METHODS: Translation was performed according to forward-backwards translation steps and transcultural adaptation. Five hundred Mandarin Chinese-speaking patients with CP were enrolled, 250 for the exploratory factor analysis (EFA) and 250 for the confirmatory factor analysis (CFA). Item analysis, reliability analysis (internal consistency, split-half reliability, test-retest reliability), and validity analysis (content validity, construct validity, and convergent validity) were performed. RESULTS: Item analysis of the Chinese version of PANQOLI revealed that the absolute t values of all items were > 3. Reliability analysis showed that Cronbach's α coefficient was 0.868, split-half coefficient was 0.934, and intraclass correlation coefficient was 0.859, demonstrating excellent reliability. For content validity, item level content validity index (I-CVI) ranged from 0.8 to 1.0, and average of I-CVI scores across all items (S-CVI/Ave) was 0.91. In construct validity analysis, EFA produced four dimensions after rotation, and results of CFA showed χ2/df = 2.346, comparative fit index (CFI) = 0.929, Tucker-Lewis index (TLI) = 0.915, and root-mean-square error of approximation (RMSEA) = 0.074. The analysis of convergent validity indicated that the Chinese version of PANQOLI was moderately correlated with the physical (r = 0.436, P < 0.001) and mental component summary (r = 0.518, P < 0.001) of the 36-Item Short Form Health Survey. CONCLUSION: The Chinese version of PANQOLI appears to be culturally appropriate, reliable, and valid for assessing the QoL amongst Chinese patients with CP.

Status quo of the public's knowledge of probiotics based on video-sharing platforms.

BACKGROUND: Probiotics have been deemed multipotent and unprecedentedly applied in the health field recently. However, there are challenges in promoting credible and reliable resources while avoiding misinformation regarding probiotics for the public. METHODS: This study analysed 400 eligible probiotic-related videos selected from YouTube, and the three most popular video-sharing platforms (Bilibili, Weibo and TikTok) in China. Video retrieval was performed on September 5th, 2022. GQS and tailored DISCERN tool assess each video's quality, usage, and reliability. A comparative analysis of videos from different sources was carried out. RESULTS: The identity distribution of probiotic video-producers was predominantly experts (n = 202, 50.50%), followed by amateurs (n = 161, 40.25%) and health-related institutions (n = 37, 9.25%). The videos' content category mainly discussed the function of probiotics (n = 120, 30%), the way to choose suitable products (n = 81, 20.25%), and the methods for taking probiotics (n = 71, 17.75%).The overall quality of videos was moderate (3/5 point) assessed by GQS, while the usage (1/6 point) and reliability (2/5 point) detailing probiotics assessed by tailored DISCERN tool were poor. The attitude of probiotic video-producers was primarily positive (n = 323, 80.75%), followed by neutral (n = 52, 13.00%) and negative (n = 25, 6.25%) (P < 0.001). CONCLUSIONS: The current study showed that videos on social media platforms publicise important information including the concepts, usage, and precautions of probiotics to the public. But the overall quality of uploaded videos about probiotics was unsatisfactory. More efforts are needed to improve the higher-quality content of probiotic-related online videos and better propagate probiotic knowledge to the public in the future.

Effects of Escherichia pollution and salinity on nutrient levels in submerged vegetated wetlands: Insights into benthic community stability and metabolisms.

Inner coastal wetland ecosystems are generally eutrophic and are often exposed to both salinity stress and Escherichia coli pollution. However, the effects of these stressors on nutrient-cycling and microbial communities are under-researched. Here, we established a vegetated wetland ecosystem in a saline environment to understand the effects of E. coli pollution on nutrient removal and benthic microorganisms. The results show that E. coli significantly inhibited nutrient removal, especially total nitrogen (TN) and ammonium (78.89-84.98 and 3.45-44.65% were removed from the non-E. coli-treated and the E. coli-treated water, respectively). Compared with non-vegetated systems, archaeal community variations at both compositional and phylogenetic levels were weakened in vegetated systems (p < 0.05). Among all the environmental factors, the ratios of PO43--P to total phosphorus and NO3--N to TN contributed the most to archaeal and bacterial community structural variations, respectively. E. coli pollution affected archaeal community succession more than bacteria (p < 0.05). E. coli also weakened the trophic transferring efficiencies between Cyanobacteria and Myxobacteria (p < 0.05). Metabolically, E. coli inhibited bacterial genetic metabolic pathways but made human infection more likely (p < 0.05). Our findings provide new insights into aquatic ecological conservation and environmental management.

Dosage of Dyrk1a shifts cells within a p21-cyclin D1 signaling map to control the decision to enter the cell cycle.

Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels.

Fully unsupervised symmetry-based mitosis detection in time-lapse cell microscopy.

MOTIVATION: Cell microscopy datasets have great diversity due to variability in cell types, imaging techniques and protocols. Existing methods are either tailored to specific datasets or are based on supervised learning, which requires comprehensive manual annotations. Using the latter approach, however, poses a significant difficulty due to the imbalance between the number of mitotic cells with respect to the entire cell population in a time-lapse microscopy sequence. RESULTS: We present a fully unsupervised framework for both mitosis detection and mother-daughters association in fluorescence microscopy data. The proposed method accommodates the difficulty of the different cell appearances and dynamics. Addressing symmetric cell divisions, a key concept is utilizing daughters' similarity. Association is accomplished by defining cell neighborhood via a stochastic version of the Delaunay triangulation and optimization by dynamic programing. Our framework presents promising detection results for a variety of fluorescence microscopy datasets of different sources, including 2D and 3D sequences from the Cell Tracking Challenge. AVAILABILITY AND IMPLEMENTATION: Code is available in github (github.com/topazgl/mitodix). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A two-dimensional ERK-AKT signaling code for an NGF-triggered cell-fate decision.

Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.

SHPRH and HLTF act in a damage-specific manner to coordinate different forms of postreplication repair and prevent mutagenesis.

Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis.

Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria.

A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)-based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART-isoquinoline and ART-quinoline hybrids showing highly improved potencies against CQ-resistant and multidrug-resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step-economic C-H activation and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid-binding protein targets of the ART-quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.

Blood in the T-tube as a side effect of hemosuccus pancreaticus.

This study examined a 60-year-old female patient with chronic pancreatitis and hemosuccus pancreaticus (HP), who was once misdiagnosed with biliary bleeding because of choledocholithiasis and T-tube drainage. Bleeding from the T-tube was demonstrated as a side effect of HP. The difficulty in diagnosing this case was attributed to the absence of pseudocysts or obvious aneurysms on CT images. Conclusively, for patients with indirect signs of biliary bleeding, such as bleeding from a T-tube, the possibility of the symptoms being side effects of pancreatic bleeding should be considered.

Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling.

Retrograde trophic signaling of nerve growth factor (NGF) supports neuronal survival and differentiation. Dysregulated trophic signaling could lead to various neurological disorders. Charcot-Marie-Tooth type 2B (CMT2B) is one of the most common inherited peripheral neuropathies characterized by severe terminal axonal loss. Genetic analysis of human CMT2B patients has revealed four missense point mutations in Rab7, a small GTPase that regulates late endosomal/lysosomal pathways, but the exact pathological mechanism remains poorly understood. Here, we show that these Rab7 mutants dysregulated axonal transport and diminished the retrograde signaling of NGF and its TrkA receptor. We found that all CMT2B Rab7 mutants were transported significantly faster than Rab7(wt) in the anterograde direction, accompanied with an increased percentile of anterograde Rab7-vesicles within axons of rat E15.5 dorsal root ganglion (DRG) neurons. In PC12M cells, the CMT2B Rab7 mutants drastically reduced the level of surface TrkA and NGF binding, presumably by premature degradation of TrkA. On the other hand, siRNA knock-down of endogenous Rab7 led to the appearance of large TrkA puncta in enlarged Rab5-early endosomes within the cytoplasm, suggesting delayed TrkA degradation. We also show that CMT2B Rab7 mutants markedly impaired NGF-induced Erk1/2 activation and differentiation in PC12M cells. Further analysis revealed that CMT2B Rab7 mutants caused axonal degeneration in rat E15.5 DRG neurons. We propose that Rab7 mutants induce premature degradation of retrograde NGF-TrkA trophic signaling, which may potentially contribute to the CMT2B disease.

Reciprocal antagonism of PIN1-APC/CCDH1 governs mitotic protein stability and cell cycle entry.

Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.

Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction.

Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single-cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the ERK-proliferation response. Altogether, our studies map the input-output relationships between ERK activity and proliferation, elucidating how heterogeneity can be generated during oncogene induction.

Reciprocal inhibition of PIN1 and APC/CCDH1 controls timely G1/S transition and creates therapeutic vulnerability.

Cyclin-dependent kinases (CDKs) mediated phosphorylation inactivates the anaphase-promoting complex (APC/CCDH1), an E3 ubiquitin ligase that contains the co-activator CDH1, to promote G1/S transition. PIN1 is a phosphorylation-directed proline isomerase and a master cancer signaling regulator. However, little are known about APC/CCDH1 regulation after phosphorylation and about PIN1 ubiquitin ligases. Here we uncover a domain-oriented reciprocal inhibition that controls the timely G1/S transition: The non-phosphorylated APC/CCDH1 E3 ligase targets PIN1 for degradation in G1 phase, restraining G1/S transition; APC/CCDH1 itself, after phosphorylation by CDKs, is inactivated by PIN1-catalyzed isomerization, promoting G1/S transition. In cancer, PIN1 overexpression and APC/CCDH1 inactivation reinforce each other to promote uncontrolled proliferation and tumorigenesis. Importantly, combined PIN1- and CDK4/6-inhibition reactivates APC/CCDH1 resulting in PIN1 degradation and an insurmountable G1 arrest that translates into synergistic anti-tumor activity against triple-negative breast cancer in vivo. Reciprocal inhibition of PIN1 and APC/CCDH1 is a novel mechanism to control timely G1/S transition that can be harnessed for synergistic anti-cancer therapy.

Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect.

BACKGROUND: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis. METHODS: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown. RESULTS: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P < 0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1ß. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA). CONCLUSION: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Alterations in the Gut Microbiota and Metabolomics of Seafarers after a Six-Month Sea Voyage.

Maintaining the health of seafarers is a difficult task during long-term voyages. Little is known about the corresponding changes in the gut microbiome-host interaction. This study recruited 30 seafarers undertaking a 6-month voyage and analyzed their gut microbiota using 16S rRNA gene sequencing. Fecal untargeted metabolomics analysis was performed using liquid chromatography-mass spectrometry. Significant changes in the composition of the gut microbiota and an increased ratio of Firmicutes/Bacteroidetes at the end (day 180) of the 6-month voyage, relative to the start (day 0), were observed. At the genus level, the abundances of Holdemanella and Plesiomonas were significantly increased, while the abundance of Bacteroides was decreased. Predicted microbial functional analysis revealed significant decreases in folate biosynthesis and biotin metabolism. Furthermore, 20 differential metabolites within six differentially enriched human metabolic pathways (including arginine biosynthesis, lysine degradation, phenylalanine metabolism, sphingolipid metabolism, pentose and glucuronate interconversions, and glycine, serine, and threonine metabolism) were identified by comparing the fecal metabolites at day 0 and day 180. Spearman correlation analysis revealed close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might affect specific human metabolic pathways. This study adopted a multi-omics approach and provides potential targets for maintaining the health of seafarers during long-term voyages. These findings are worthy of more in-depth exploration in future studies. IMPORTANCE Maintaining the health of seafarers undertaking long-term voyages is a difficult task. Apart from the alterations in the gut microbiome and fecal metabolites after a long-term voyage, our study also revealed that 20 differential metabolites within six differentially enriched human metabolic pathways are worthy of attention. Moreover, we found close relationships between the 14 differential microbiota members and the six differential fecal metabolites that might impact specific human metabolic pathways. Accordingly, preventative measures, such as adjusting the gut microbiota by decreasing potential pathobionts or increasing potential probiotics as well as offsetting the decrease in B vitamins and beneficial metabolites (e.g., d-glucuronic acid and citrulline) via dietary adjustment or nutritional supplements, might improve the health of seafarers during long-term sea voyages. These findings provide valuable clues about gut microbiome-host interactions and propose potential targets for maintaining the health of seafarers engaged in long-term sea voyages.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA.

BACKGROUND: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood. METHODS: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays. RESULTS: We developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photo-crosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods. CONCLUSIONS: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase.

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors induce apoptosis in a variety of tissues; however, the mechanism underlying this induction is largely unknown. Here, we demonstrate that TGF-beta induces the expression of the death-associated protein kinase (DAP-kinase) as an immediate early response in cells that undergo apoptosis in response to TGF-beta. DAP-kinase is a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that the DAP-kinase promoter is activated by TGF-beta through the action of Smad2, Smad3 and Smad4. Overexpression of DAP-kinase triggers apoptosis in the absence of TGF-beta, whereas inhibition of DAP-kinase activity protects cells from TGF-beta-induced apoptosis, blocks TGF-beta-induced release of cytochrome c from mitochondria and prevents TGF-beta-induced dissipation of the mitochondrial membrane potential. Our findings indicate that DAP-kinase mediates TGF-beta-dependent apoptosis by linking Smads to mitochondrial-based pro-apoptotic events.