National Center of Biomedical Analysis

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.

Cancer cells are known to undergo metabolic reprogramming to sustain survival and rapid proliferation, however, it remains to be fully elucidated how oncogenic lesions coordinate the metabolic switch under various stressed conditions. Here we show that deprivation of glucose or glutamine, two major nutrition sources for cancer cells, dramatically activated serine biosynthesis pathway (SSP) that was accompanied by elevated cMyc expression. We further identified that cMyc stimulated SSP activation by transcriptionally upregulating expression of multiple SSP enzymes. Moreover, we demonstrated that SSP activation facilitated by cMyc led to elevated glutathione (GSH) production, cell cycle progression and nucleic acid synthesis, which are essential for cell survival and proliferation especially under nutrient-deprived conditions. We further uncovered that phosphoserine phosphatase (PSPH), the final rate-limiting enzyme of the SSP pathway, is critical for cMyc-driven cancer progression both in vitro and in vivo, and importantly, aberrant expression of PSPH is highly correlated with mortality in hepatocellular carcinoma (HCC) patients, suggesting a potential causal relation between this cMyc-regulated enzyme, or SSP activation in general, and cancer development. Taken together, our results reveal that aberrant expression of cMyc leads to the enhanced SSP activation, an essential part of metabolic switch, to facilitate cancer progression under nutrient-deprived conditions.

Calcium is thought to play an important role in regulating mitochondrial function. Evidence suggests that an increase in mitochondrial calcium can augment ATP production by altering the activity of calcium-sensitive mitochondrial matrix enzymes. In contrast, the entry of large amounts of mitochondrial calcium in the setting of ischemia-reperfusion injury is thought to be a critical event in triggering cellular necrosis. For many decades, the details of how calcium entered the mitochondria remained a biological mystery. In the past few years, significant progress has been made in identifying the molecular components of the mitochondrial calcium uniporter complex. Here, we review how calcium enters and leaves the mitochondria, the growing insight into the topology, stoichiometry and function of the uniporter complex, and the early lessons learned from some initial mouse models that genetically perturb mitochondrial calcium homeostasis.

The cytosolic pattern recognition receptor (PRR) NOD-like receptor family, pyrin domain containing 3 (NLRP3) senses a wide range of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Upon activation, NLRP3 triggers the assembly of inflammasome via the self-oligomerization and the recruitment of apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and pro-caspase-1, facilitating the robust immune responses including the secretion of proinflammatory cytokines and pyroptosis. The NLRP3 inflammasome must be well orchestrated to prevent the aberrant activations under physiological and pathological conditions, because uncontrolled activation of NLRP3 inflammasome is one of the major causes of a variety of autoimmune diseases and metabolic disorders. Therefore, understanding the molecular mechanisms for controlling NLRP3 inflammasome activation may provide novel strategies for the treatment of NLRP3-related diseases. Although NLRP3 inflammasome can be regulated at the transcriptional level, the post-translational modification (PTM) of NLRP3 as well as other inflammasome components has also been showed to be critical for the regulation of its activation. Several kinases and phosphatases have been shown to control NLRP3 inflammasome activation in response to either exogenous pathogen infections or endogenous molecules, such as bile acids. In this review, we summarize our current knowledge of phosphorylation patterns and their functional role in the regulation of NLRP3 inflammasome, and suggest interesting areas for future research.

Drastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon- and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.

Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Neural stem cells (NSCs) in the adult mammalian central nervous system (CNS) hold the key to neural regeneration through proper activation, differentiation, and maturation, to establish nascent neural networks, which can be integrated into damaged neural circuits to repair function. However, the CNS injury microenvironment is often inhibitory and inflammatory, limiting the ability of activated NSCs to differentiate into neurons and form nascent circuits. Here we report that neurotrophin-3 (NT3)-coupled chitosan biomaterial, when inserted into a 5-mm gap of completely transected and excised rat thoracic spinal cord, elicited robust activation of endogenous NSCs in the injured spinal cord. Through slow release of NT3, the biomaterial attracted NSCs to migrate into the lesion area, differentiate into neurons, and form functional neural networks, which interconnected severed ascending and descending axons, resulting in sensory and motor behavioral recovery. Our study suggests that enhancing endogenous neurogenesis could be a novel strategy for treatment of spinal cord injury.

Medical imaging denoising faces great challenges, yet is in great demand. With its distinctive characteristics, medical imaging denoising in the image domain requires innovative deep learning strategies. In this study, we propose a simple yet effective strategy, the content-noise complementary learning (CNCL) strategy, in which two deep learning predictors are used to learn the respective content and noise of the image dataset complementarily. A medical image denoising pipeline based on the CNCL strategy is presented, and is implemented as a generative adversarial network, where various representative networks (including U-Net, DnCNN, and SRDenseNet) are investigated as the predictors. The performance of these implemented models has been validated on medical imaging datasets including CT, MR, and PET. The results show that this strategy outperforms state-of-the-art denoising algorithms in terms of visual quality and quantitative metrics, and the strategy demonstrates a robust generalization capability. These findings validate that this simple yet effective strategy demonstrates promising potential for medical image denoising tasks, which could exert a clinical impact in the future. Code is available at: https://github.com/gengmufeng/CNCL-denoising.

Abstract Purpose: The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3′ region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC. Experimental Design: The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non–small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver. Results: Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1). Conclusions: This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584–ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.

Two hallmarks for cancer cells are the accelerated cell cycle progression as well as the altered metabolism, however, how these changes are coordinated to optimize the growth advantage for cancer cells are still poorly understood. Here we identify that Polo-like kinase 1 (Plk1), a key regulator for cell mitosis, plays a critical role for biosynthesis in cancer cells through activating pentose phosphate pathway (PPP). We find that Plk1 interacts with and directly phosphorylates glucose-6-phosphate dehydrogenase (G6PD). By activating G6PD through promoting the formation of its active dimer, Plk1 increases PPP flux and directs glucose to the synthesis of macromolecules. Importantly, we further demonstrate that Plk1-mediated activation of G6PD is critical for its role to promote cell cycle progression and cancer cell growth. Collectively, these findings establish a critical role for Plk1 in regulating biosynthesis in cancer cells, exemplifying how cell cycle progression and metabolic reprogramming are coordinated for cancer progression.

Polycystic ovary syndrome (PCOS) is a common, clinically heterogeneous endocrine disorder affecting women of reproductive age, associated with endocrinopathy and metabolic abnormalities. Although some metabolic parameters have been investigated, very little information has been reported on the changes of small metabolites in biofluids. The aim of this study was to establish the metabolic profile of PCOS and compare it with that of controls. In this cross-sectional study of 34 women with PCOS and 36 controls, contents of small metabolites and lipids in plasma samples were measured using nuclear magnetic resonance (NMR)-based techniques and analyzed using multivariate statistical methods. Significant decrease (P < 0.05) in the levels of amino acids (leucine, isoleucine, methionine, glutamine, and arginine), citrate, choline, and glycerophosphocholine/phosphocholine (GPC/PC), and increase (P < 0.05) in the levels of lactate, dimethylamine (DMA), creatine, and N-acetyl glycoproteins were observed in PCOS patients compared with the controls. Subgroups of patients with obesity, metabolic syndrome, or hyperandrogenism exhibited greater metabolic deviations than their corresponding subgroups without these factors. PCOS patients have perturbations in amino acid metabolism, the tricarboxylic acid (TCA) cycle, and gut microflora, as well as mild disturbances in glucose and lipid metabolism. The elevated level of N-acetyl glycoproteins demonstrates the existence of low-grade chronic inflammation in PCOS patients.

Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. SIGNIFICANCE: β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Hepatocellular carcinoma (HCC) is a common malignancy that has region specific etiologies. Unfortunately, 85% of cases of HCC are diagnosed at an advanced stage. Reliable biomarkers for the early diagnosis of HCC are urgently required to reduced mortality and therapeutic expenditure. We established a non-targeted gas chromatography-time of flight-mass spectrometry (GC-TOFMS) metabolomics method in conjunction with Random Forests (RF) analysis based on 201 serum samples from healthy controls (NC), hepatitis B virus (HBV), liver cirrhosis (LC) and HCC patients to explore the metabolic characteristics in the progression of hepatocellular carcinogenesis. Ultimately, 15 metabolites were identified intimately associated with the process. Phenylalanine, malic acid and 5-methoxytryptamine for HBV vs. NC, palmitic acid for LC vs. HBV, and asparagine and β-glutamate for HCC vs. LC were screened as the liver disease-specific potential biomarkers with an excellent discriminant performance. All the metabolic perturbations in these liver diseases are associated with pathways for energy metabolism, macromolecular synthesis, and maintaining the redox balance to protect tumor cells from oxidative stress.

Although immune checkpoint blockade have demonstrated promising results, their effects on gastric cancer (GC) are under investigation. Understanding the clinical significance of PD1 and its ligands' expression, together with T cell infiltration might provide clues for biomarkers screening in GC immunotherapy. Immunohistochemistry were performed on a tissue microarray including 1,014 GC specimens using PD1, PDL1 and PDL2 antibodies. T cell markers CD3 and CD8 were also stained and quantified by automated image analysis. Correlation with clinical features and outcome were analyzed after controlling for potential confounders including EBV infection, HER2, C-met and PCNA expression. 37.8% of the cases showed membranous PD-L1 expression in tumor cells and 74.9% in infiltrating immune cells. PDL1 expression rate was rather higher in patients without metastasis, in EBV positive group and those with C-met and PCNA expression. GC patients with high level PDL1 expression exhibited better survival. GC Patients with higher T cell infiltration also showed elevated PDL1, PDL2 and PD1 expression and predict favorable outcome, indicating an adaptive immune resistance mechanism may exist. The group of patients infiltrated with lower density CD3+ T cells also without PDL1 expression in tumor cells predict the worst outcome in the subgroup of different PTNM stage, which may suggest an inactive immune status. These results highlights the need to assess both PDL1 expression in all tumor context and the characterization of the GC immune microenvironment.

Low levels of reactive oxygen species (ROS) are crucial for maintaining cancer stem cells (CSCs) and their ability to resist therapy, but the ROS regulatory mechanisms in CSCs remains to be explored. Here, we discover that prohibitin (PHB) specifically regulates mitochondrial ROS production in glioma stem-like cells (GSCs) and facilitates GSC radiotherapeutic resistance. We find that PHB is upregulated in GSCs and is associated with malignant gliomas progression and poor prognosis. PHB binds to peroxiredoxin3 (PRDX3), a mitochondrion-specific peroxidase, and stabilizes PRDX3 protein through the ubiquitin-proteasome pathway. Knockout of PHB dramatically elevates ROS levels, thereby inhibiting GSC self-renewal. Importantly, deletion or pharmacological inhibition of PHB potently slows tumor growth and sensitizes tumors to radiotherapy, thus providing significant survival benefits in GSC-derived orthotopic tumors and glioblastoma patient-derived xenografts. These results reveal a selective role of PHB in mitochondrial ROS regulation in GSCs and suggest that targeting PHB improves radiotherapeutic efficacy in glioblastoma.

DNA damage response (DDR) acts as a tumorigenesis barrier, and any defects in the DDR machinery may lead to cancer. SOX4 expression is elevated in many types of tumors; however, its role in DDR is still largely unknown. Here, we show that SOX4, a new DNA damage sensor, is required for the activation of p53 tumor suppressor in response to DNA damage. Notably, SOX4 interacts with and stabilizes p53 protein by blocking Mdm2-mediated p53 ubiquitination and degradation. Furthermore, SOX4 enhances p53 acetylation by interacting with p300/CBP and facilitating p300/CBP/p53 complex formation. In concert with these results, SOX4 promotes cell cycle arrest and apoptosis, and it inhibits tumorigenesis in a p53-dependent manner. Therefore, these findings highlight SOX4 as a potential key factor in regulating DDR-associated cancer.

The 66 kDa estrogen receptor alpha (ERα66) is the main molecular target for endocrine therapy such as tamoxifen treatment. However, many patients develop resistance with unclear mechanisms. In a large cohort study of breast cancer patients who underwent surgery followed by tamoxifen treatment, we demonstrate that ERα36, a variant of ERα66, correlates with poor prognosis. Mechanistically, tamoxifen directly binds and activates ERα36 to enhance the stemness and metastasis of breast cancer cells via transcriptional stimulation of aldehyde dehydrogenase 1A1 (ALDH1A1). Consistently, the tamoxifen-induced stemness and metastasis can be attenuated by either ALDH1 inhibitors or a specific ERα36 antibody. Thus, tamoxifen acts as an agonist on ERα36 in breast cancer cells, which accounts for hormone therapy resistance and metastasis of breast cancer. Our study not only reveals ERα36 as a stratifying marker for endocrine therapy but also provides a promising therapeutic avenue for tamoxifen-resistant breast cancer.

Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community.

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.