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When using selective laser sintering to print parts with thin-walled structures, the thermal action of the laser can cause thermal stresses that lead to plastic deformation, resulting in large warpage and dimensional deviations. To address this issue, this study proposes a bottom support method for selective laser sintering. The impact of lattice-type, concentric-type, and cross-type support structures with varying filling densities and thicknesses on the suppression of warpage and dimensional errors was investigated. The optimal process parameters for each support structure were then determined through optimization. The findings of this study demonstrated a reduction in Z-axis dimensional errors of the workpiece following the addition of supports. The reduction amounted to 33.809%, 86.160%, and 66.214%, respectively, compared to the original workpiece. Moreover, the corresponding warpage was reduced by 35.673%, 46.189%, and 46.059% for each respective case, showcasing an improvement in the printing precision. Therefore, the bottom support effectively reduces dimensional and shape errors in thin-walled parts printed by selective laser sintering. Specifically, the results obtained indicated that the concentric type of support is more effective in reducing dimensional errors and enhancing the shape accuracy of the printed workpiece. Conversely, the cross type of support demonstrated superior capabilities in minimizing the consumption of printing materials while still delivering satisfactory results. Thus, this study holds promise for contributing to the advancement of thin-walled part quality using selective laser sintering technology. This research can contribute to achieving greater accuracy in the fabrication of parts through 3D printing.

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation “alarmone” (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response. However, the function of MESH1 remains unknown until our recent discovery of MESH1 as the first cytosolic NADPH phosphatase that regulates ferroptosis. To further understand whether MESH1 knockdown triggers a similar transcriptional response in mammalian cells, here, we employed RNA-Seq to analyze the transcriptome response to MESH1 knockdown in human cancer cells. We find that MESH1 knockdown induced different genes involving endoplasmic reticulum (ER) stress, especially ATF3, one of the ATF4-regulated genes in the integrative stress responses (ISR). Furthermore, MESH1 knockdown increased ATF4 protein, eIF2a phosphorylation, and induction of ATF3, XBPs, and CHOP mRNA. ATF4 induction contributes to ~30% of the transcriptome induced by MESH1 knockdown. Concurrent ATF4 knockdown re-sensitizes MESH1-depleted RCC4 cells to ferroptosis, suggesting its role in the ferroptosis protection mediated by MESH1 knockdown. ATF3 induction is abolished by the concurrent knockdown of NADK, implicating a role of NADPH accumulation in the integrative stress response. Collectively, these results suggest that MESH1 depletion triggers ER stress and ISR as a part of its overall transcriptome changes to enable stress survival of cancer cells. Therefore, the phenotypic similarity of stress tolerance caused by MESH1 removal and NADPH accumulation is in part achieved by ISR to regulate ferroptosis.

Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosis-inducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of “alarmone” (p)ppGpp to arrest proliferation and reprogram transcriptome. While mammalian genomes encode MESH1—the homolog of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumor sphere formation, and retarded xenograft growth. These antitumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed expression of TAZ, a HIPPO coactivator, and proliferative gene. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, reduced sphere formation, tumor growth inhibition, dNTP depletion, and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.

Background & aims The outcome of liver injury is dictated by factors that control the accumulation of myofibroblastic (activated) hepatic stellate cells (MF-HSCs) but therapies that specifically block this process have not been discovered. We evaluated the hypothesis that MF-HSCs and liver fibrosis could be safely reduced by inhibiting the cysteine/glutamate antiporter xCT. Methods xCT activity was disrupted in both HSC lines and primary mouse HSCs to determine its effect on HSC biology. For comparison, xCT expression and function were also determined in primary mouse hepatocytes. Finally, the roles of xCT were assessed in mouse models of liver fibrosis. Results We found that xCT mRNA levels were almost a log-fold higher in primary mouse HSCs than in primary mouse hepatocytes. Further, primary mouse HSCs dramatically induced xCT as they became MF, and inhibiting xCT blocked GSH synthesis, reduced growth and fibrogenic gene expression and triggered HSC ferroptosis. Doses of xCT inhibitors that induced massive ferroptosis in HSCs had no effect on hepatocyte viability in vitro, and xCT inhibitors reduced liver fibrosis without worsening liver injury in mice with acute liver injury. However, TGFβ treatment up-regulated xCT and triggered ferroptosis in cultured primary mouse hepatocytes. During chronic liver injury, xCT inhibitors exacerbated injury, impaired regeneration and failed to improve fibrosis, confirming that HSCs and hepatocytes deploy similar mechanisms to survive chronic oxidative stress. Conclusions Inhibiting xCT can suppress myofibroblastic activity and induce ferroptosis of MF-HSCs. However, targeting xCT inhibition to MF-HSCs will be necessary to exploit ferroptosis as an anti-fibrotic strategy.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically reexpressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo necroptosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

Background and AimsLiver fibrosis develops in the context of excessive oxidative stress, cell death and accumulation of myofibroblasts (MFs) derived from hepatic stellate cells (HSCs). Ferroptosis is a type of regulated cell death that can be caused by inhibiting the cystine/glutamate antiporter xCT. However, while xCT is induced in various liver diseases, its role in HSC activation and liver fibrosis is unknown. We hypothesized that xCT is required for HSCs to antagonize ferroptosis and remain myofibroblastic.MethodsxCT activity was disrupted by siRNA or pharmacological inhibitors in MF-HSC cell lines to determine its effect on redox homeostasis, growth, myofibroblastic activity and viability. xCT expression was then determined by RNA sequencing and RT-PCR during primary HSC activation, and its role in HSC trans-differentiation was assessed. For comparison, xCT expression and function were also determined in primary hepatocytes. Finally, the roles of xCT in HSC accumulation and liver fibrosis were assessed in mice treated acutely with CCl4.ResultsInhibiting xCT in MF-HSCs decreased intracellular glutathione (GSH), suppressed growth and fibrogenesis, and induced cell death. These effects were rescued by antioxidants, an iron chelator, and a canonical ferroptosis inhibitor, but not by inhibitors of apoptosis or necrosis. xCT was dramatically up-regulated during primary HSC activation, and inhibiting xCT suppressed myofibroblastic trans-differentiation and induced ferroptosis. In contrast, healthy hepatocytes were relatively insensitive to ferroptosis induced by xCT inhibition. In vivo, inhibiting xCT systemically reduced MF-HSC accumulation and liver fibrosis after a single dose of CCl4 without exacerbating liver injury or reducing hepatocyte regeneration.ConclusionCompared to healthy hepatocytes, MF-HSCs are exquisitely sensitive to ferroptosis induced by inhibiting xCT. In acutely injured livers, systemic inhibitors of xCT can inhibit fibrosis without worsening liver injury. Further research is needed to determine if this therapeutic window remains sufficiently robust to safely target MF-HSCs and inhibit fibrogenesis in chronically injured liver.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically re-expressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo programmed necrosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

Nutrient deprivation triggers stringent response in bacteria, allowing rapid reallocation of resources from proliferation toward stress survival. Critical to this process is the accumulation/degradation of (p)ppGpp regulated by the RelA/SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Therefore, the function of MESH1 remains a mystery. Here, we report that human MESH1 is an efficient cytosolic NADPH phosphatase, an unexpected enzymatic activity that is captured by the crystal structure of the MESH1-NADPH complex. MESH1 depletion promotes cell survival under ferroptosis-inducing conditions by sustaining the level of NADPH, an effect that is reversed by the simultaneous depletion of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Importantly, MESH1 depletion also triggers extensive transcriptional changes that are distinct from the canonical integrated stress response but resemble the bacterial stringent response, implicating MESH1 in a previously uncharacterized stress response in mammalian cells.

Additional file 1: Figure S1. Restoration of TDAG8 gene expression results in increased phosphorylation of CREB at serine 133 in U937 and Ramos cells. (A) Restoration of TDAG8 gene expression in U937 cells results in stimulation of CREB phosphorylation at serine 133 indicating the TDAG8 activity level is increased. (B) TDAG8 gene expression is restored in Ramos cells to a level that is physiologically relevant. (C) Restoration of TDAG8 gene expression in Ramos cells results in stimulation of CREB phosphorylation at serine 133 indicating the TDAG8 activity level is increased. **P 0.05, *P 0.05, *P 0.05, *P 0.05, *P

Summary: Despite recent advances, the poor outcomes in renal cell carcinoma (RCC) suggest novel therapeutics are needed. Ferroptosis is a form of regulated cell death, which may have therapeutic potential toward RCC; however, much remains unknown about the determinants of ferroptosis susceptibility. We found that ferroptosis susceptibility is highly influenced by cell density and confluency. Because cell density regulates the Hippo-YAP/TAZ pathway, we investigated the roles of the Hippo pathway effectors in ferroptosis. TAZ is abundantly expressed in RCC and undergoes density-dependent nuclear or cytosolic translocation. TAZ removal confers ferroptosis resistance, whereas overexpression of TAZS89A sensitizes cells to ferroptosis. Furthermore, TAZ regulates the expression of Epithelial Membrane Protein 1 (EMP1), which, in turn, induces the expression of nicotinamide adenine dinucleotide phosphate (NADPH) Oxidase 4 (NOX4), a renal-enriched reactive oxygen species (ROS)-generating enzyme essential for ferroptosis. These findings reveal that cell density-regulated ferroptosis is mediated by TAZ through the regulation of EMP1-NOX4, suggesting its therapeutic potential for RCC and other TAZ-activated tumors. : Yang et al. show that ferroptosis sensitivity in renal cell carcinoma (RCC) is regulated by cell density through the TAZ-EMP1-NOX4 pathway. These findings reveal TAZ as a genetic determinant of ferroptosis in RCC. In addition, ferroptosis may hold therapeutic potential for RCC and other TAZ-activated tumors. Keywords: cell density, ferroptosis, renal cell carcinoma, erastin, Hippo pathway, WW Domain Containing Transcription Regulator 1, TAZ, Epithelial Membrane Protein 1, EMP1, NADPH Oxidase 4, NOX4

The capacity of tumor cells to survive various stresses is an essential property that endows them invulnerability. One of the bacterial key adaptive strategies against nutrient deprivation is stringent response. In bacteria, nutrient deprivation leads to an elevation of a signaling alarmone, guanosine tetraphosphate ((p)ppGpp) to repress the protein synthesis and proliferation activity in order to maintain cell viability. The accumulation of (p)ppGpp is partially caused by the repression of its hydrolase, SpoT. However, this strategy still remains elusive in humans, but the human ortholog of SpoT-MESH1 has been discovered. MESH1 also has the hydrolase activity on (p)ppGpp in vitro, but human cells lack the required alarmone (p)ppGpp and its synthetase, which restricts the discovery of stringent response in humans. Unexpectedly, our recent data suggests that MESH1 depletion allows cell survival under cystine deprivation, an extremely oxidative stress, yet with a slow proliferation rate. These correspond to bacterial stringent response phenotypes. Moreover, our gene expression profiling shows that MESH1 silencing triggers a global transcriptional reprogramming with a similarity to bacterial transcriptional responses of stringent response. Altogether, these evidences inspire us to speculate on the existence of human stringent response with similar cell protective functions, albeit in the absence of (p)ppGpp. In this study, I aim to identify the molecular pathways and regulatory mechanisms that MESH1 adopts to explain the MESH1-related stringent response phenotypes in human cancer cell lines. One interesting phenomenon we observed from our genome-wide RNA screen is that MESH1 silencing significantly represses TAZ RNA level. TAZ serves as a key co-activator involved in the HIPPO pathway that has been well studied to regulate cell proliferation, cell stemness, and etc, which are perfectly relevant to the MESH1-related stringent response phenotypes. Therefore, I will specifically determine the functional roles of TAZ repression upon MESH1 removal in the expression of stringent response features by genetic tools. Furthermore, I will characterize the transcriptional control of TAZ and other gene signatures of human stringent response. We observed that MESH1 depletion significantly represses the acetyl-CoA:CoA ratio and further inhibits the histone acetylation activity that presumably turns off/on certain gene transcription. Based on these facts, I will determine the importance of histone modification on the transcriptional reprogramming upon MESH1 silencing. Successful completion of this study will equip us with more thorough understandings of stress response mechanisms in humans so that we can improve the treatment for stress-related diseases, such as cancer, by providing a novel therapeutic target, MESH1. Note: This abstract was not presented at the meeting. Citation Format: Tianai Sun, Chien-Kuang Ding, Jen-Tsan A. Chi. Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2667.

Biological diversity on Earth depends on the multiplication of species or speciation, which is the evolution of reproductive isolation such as hybrid sterility between two new species. An unsolved puzzle is the exact mechanism(s) that causes two genomes to diverge from their common ancestor so that some divergent genes no longer function properly in the hybrids. Here we report genetic analyses of divergent genes controlling male fertility and sex ratio in two very young fruitfly species, Drosophila albomicans and D. nasuta. A majority of the genetic divergence for both traits is mapped to the same regions by quantitative trait loci mappings. With introgressions, six major loci are found to contribute to both traits. This genetic colocalization implicates that genes for hybrid male sterility have evolved primarily for controlling sex ratio. We propose that genetic conflicts over sex ratio may operate as a perpetual dynamo for genome divergence. This particular evolutionary mechanism may largely contribute to the rapid evolution of hybrid male sterility and the disproportionate enrichment of its underlying genes on the X chromosome – two patterns widely observed across animals., Author Summary Millions of species live on Earth, thanks to an evolutionary process that splits one species to two or more new species. The formation of new species is benchmarked by the evolution of reproductive isolation (RI) such as hybrid sterility between new species. The fundamental question of how RI evolves, however, remains largely unknown. In a pair of very young fruitfly species, we localized six loci expressing dual functions of hybrid male sterility (HMS) and sex ratio distortion, implicating an evolutionary causal link between these two traits. The rapid evolution of HMS widely observed across animal taxa can be attributed to the rapid evolution of genes controlling sex chromosome segregation. All genes in a genome are not equal. This study suggests that conflicts among various parts of a genome might confer strong evolutionary pressure—a mechanism that has hitherto been regarded as rare and could actually be more ubiquitous than currently appreciated.

Additional file 2: Table S1. TDAG8 gene expression in various cancer types compared to normal tissues.

Additional file 2: Table S1. TDAG8 gene expression in various cancer types compared to normal tissues.