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While most respiratory viral infections resolve with little harm to the host, severe symptoms arise when infection triggers an aberrant inflammatory response that damages lung tissue. Host regulators of virally induced lung inflammation have not been well defined. Here, we show that enrichment for sialylated, but not asialylated immunoglobulin G (IgG), predicted mild influenza disease in humans and was broadly protective against heterologous influenza viruses in a murine challenge model. Mechanistic studies show that sialylated IgG mediated this protection by inducing the transcription factor repressor element-1 silencing transcription factor (REST), which repressed nuclear factor κB (NF-κB)-driven responses, preventing severe lung inflammation and protecting lung function during influenza infection. Therapeutic administration of a recombinant, sialylated Fc molecule in clinical development similarly activated REST and protected against severe influenza disease, demonstrating that this pathway could be clinically harnessed. Overall, induction of REST through sialylated IgG signaling is a strategy to limit inflammatory disease sequelae in infections caused by antigenically distinct influenza strains., Competing Interests: Declaration of interests T.T.W. is a scientific advisor for Nuvig Therapeutics. M.S.D. is a consultant or on the Scientific Advisory Board for Inbios, Vir Biotechnology, Topspin Therapeutics, Akagera Medicines, Merck, GlaxoSmithKline, and Moderna. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Emergent BioSolutions, Moderna, Topspin Therapeutics, and Vir Biotechnology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multiple cancers, including colorectal cancer (CRC), are more frequent and often more aggressive in individuals with obesity. Here, we showed that macrophages accumulated within tumors of patients with obesity and CRC and in obese CRC mice and that they promoted accelerated tumor growth. These changes were initiated by oleic acid accumulation and subsequent tumor cell-derived acid production and were driven by macrophage signaling through the acid-sensing receptor GPR65. We found a similar role for GPR65 in hepatocellular carcinoma (HCC) in obese mice. Tumors in patients with obesity and CRC or HCC also exhibited increased GPR65 expression, suggesting that the mechanism revealed here may contribute to tumor growth in a range of obesity-associated cancers and represent a potential therapeutic target.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development 1,2 , and increased stiffness is known to promote HCC progression in cirrhotic conditions 3,4 . Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic β-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-β1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Hypoxia-induced adenosine creates an immunosuppressive tumor microenvironment (TME) and dampens the efficacy of immune checkpoint inhibitors (ICIs). We found that hypoxia-inducible factor 1 (HIF-1) orchestrates adenosine efflux through two steps in hepatocellular carcinoma (HCC). First, HIF-1 activates transcriptional repressor MXI1, which inhibits adenosine kinase (ADK), resulting in the failure of adenosine phosphorylation to adenosine monophosphate. This leads to adenosine accumulation in hypoxic cancer cells. Second, HIF-1 transcriptionally activates equilibrative nucleoside transporter 4, pumping adenosine into the interstitial space of HCC, elevating extracellular adenosine levels. Multiple in vitro assays demonstrated the immunosuppressive role of adenosine on T cells and myeloid cells. Knockout of ADK in vivo skewed intratumoral immune cells to protumorigenic and promoted tumor progression. Therapeutically, combination treatment of adenosine receptor antagonists and anti-PD-1 prolonged survival of HCC-bearing mice. We illustrated the dual role of hypoxia in establishing an adenosine-mediated immunosuppressive TME and offered a potential therapeutic approach that synergizes with ICIs in HCC.

Background and Aims: Prognosis of HCC remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found that polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC., Approach and Results: An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation, and senescence. The cytosolic DNA accumulation elicited the DEAD box helicase 41-stimulator of interferon genes-interferon regulatory factor 3/7-NF-κβ cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes, which recruit immune cells. CFI-400945 was evaluated in liver-specific p53/phosphatase and tensin homolog knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of cluster of differentiation 4-positive (CD4 + ), CD8 + T cells, macrophages, and natural killer cells. Combination therapy of CFI-400945 with anti-programmed death-1 showed a tendency to improve HCC survival., Conclusions: We show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing antitumor immunity to achieve a durable suppressive effect even in late-stage mouse HCC., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B 4 , which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer., Competing Interests: Declaration of interests I.L.L., T.R.P., and E.G.E. are co-inventors on a patent application filed by Stanford University related to this work. W.-C.S. owns equity in, receives a consultant fee and research grant from, and is an inventor of patents licensed to Kira Pharmaceuticals and Aevitas Therapeutics. The other authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background & Aims: Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs., Methods: A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing., Results: Based on the CD8 + T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53 KO /MYC OE ) and a hot tumor (Keap1 KO /MYC OE ) were further characterized. Tumor-infiltrating CD8 + T cells from Keap1 KO /MYC OE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8 + T-cell phenotype compared to those in Trp53 KO /MYC OE HCCs. The TKI sorafenib sensitized Trp53 KO /MYC OE HCCs to anti-PD-1 treatment., Conclusion: Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors., Impact and Implications: Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8 + T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8 + T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Deregulation of cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells could bypass this checkpoint mechanism. In this study, we showed the clinical relevance of threonine tyrosine kinase (TTK) protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here, we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth and induced highly aneuploid HCC cells, DNA damage, and micronuclei formation. We identified that CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4 + T cells, and CD8 + T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O 2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive., (© 2022. The Author(s).)

Liver cancers consist primarily of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Immune checkpoint inhibitors have emerged as promising therapeutic agents against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells that interacts with its ligand programmed death-ligand 1 (PD-L1) found on cancer cells. Blocking PD-1/PD-L1 binding improves T-cell survival, proliferation and cytotoxicity, which enhances their antitumor activity. Better understanding of the molecular mechanisms governing PD-1/PD-L1 response is essential to the development of predictive markers and therapeutic combinations that could improve the efficiency of anti-PD-1/PD-L1 treatment. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) has been recently identified as a major regulator of PD-L1. Another member in the CMTM family, CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4), has been shown to compensate for the effects of CMTM6 when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. Up-regulated CMTM4 in patients with HCC and ICC is associated with poor patient survival, potentially due to its function in stabilizing PD-L1 expression, hence facilitating escape from T cell-mediated cytotoxicity. We confirmed the role of CMTM4 as a positive regulator of PD-L1 in multiple HCC and ICC cell lines and demonstrated that CMTM4 stabilizes PD-L1 through posttranslational mechanisms. In vivo, suppression of Cmtm4 inhibited HCC growth and increased CD8 + T-cell infiltration in immunocompetent mice. Furthermore, we found that depletion of CMTM4 sensitized HCC tumor to anti-PD-L1 treatment compared with control. This suggests that CMTM4 expression level could be a predictive marker for patient response to anti-PD-L1 treatment, and CMTM4 depletion can potentially be used to enhance the clinical benefits of anti-PD-L1 immunotherapy in patients with liver cancer., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation, and differentiation. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein Angiomotin (Amot) inhibits migration and expansion of the physiological and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds Talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is an important component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extracellular matrix to form a functional vascular network., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Aims: HCC undergoes active metabolic reprogramming. Reactive oxygen species (ROS) are excessively generated in cancer cells and are neutralized by NADPH. Malic enzymes (MEs) are the less studied NADPH producers in cancer., Approach and Results: We found that ME1, but not ME3, was regulated by the typical oxidative stress response pathway mediated by kelch-like ECH associated protein 1/nuclear factor erythroid 2-related factor (NRF2). Surprisingly, ME3 was constitutively induced by superenhancers. Disruption of any ME regulatory pathways decelerated HCC progression and sensitized HCC to sorafenib. Therapeutically, simultaneous blockade of NRF2 and a superenhancer complex completely impeded HCC growth. We show that superenhancers allow cancer cells to counteract the intrinsically high level of ROS through constitutively activating ME3 expression. When HCC cells encounter further episodes of ROS insult, NRF2 allows cancer cells to adapt by transcriptionally activating ME1., Conclusions: Our study reveals the complementary regulatory mechanisms which control MEs and provide cancer cells multiple layers of defense against oxidative stress. Targeting both regulatory mechanisms represents a potential therapeutic approach for HCC treatment., (© 2021 by the American Association for the Study of Liver Diseases.)

Hypoxia, low oxygen (O 2 ), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1β as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Immune checkpoint inhibitors are effective in the treatment of some hepatocellular carcinomas (HCCs), but these tumors do not always respond to inhibitors of programmed cell death 1 (PDCD1, also called PD1). We investigated mechanisms of resistance of liver tumors in mice to infiltrating T cells., Methods: Mice were given hydrodynamic tail vein injections of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) and transposon vectors to disrupt Trp53 and overexpress C-Myc (Trp53 KO /C-Myc OE mice). Pvrl1 and Pvrl3 were knocked down in Hepa1-6 cells by using short hairpin RNAs. Hepa1-6 cells were injected into livers of C57BL/6 mice; some mice were given intraperitoneal injections of antibodies against PD1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or CD8 before the cancer cells were injected. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction; tumors were analyzed by mass cytometry using markers to detect T cells and other lymphocytes. We obtained HCC and nontumorous liver tissues and clinical data from patients who underwent surgery in Hong Kong and analyzed the tissues by immunohistochemistry., Results: Trp53 KO /C-Myc OE mice developed liver tumors in 3-5 weeks; injections of anti-PD1 did not slow tumor development. Tumors from mice given anti-PD1 had larger numbers of memory CD8 + T cells (CD44 + CD62L - KLRG1 int ) and T cells that expressed PD1, lymphocyte activating 3 (LAG3), and TIGIT compared with mice not given the antibody. HCC tissues from patients had higher levels of PVRL1 messenger RNA and protein than nontumorous tissues. Increased PVRL1 was associated with shorter times of disease-free survival. Knockdown of Pvrl1 in Hepa1-6 cells caused them to form smaller tumors in mice, infiltrated by higher numbers of CD8 + T cells that expressed the inhibitory protein TIGIT; these effects were not observed in mice with depletion of CD8 + T cells. In Hepa1-6 cells, PVRL1 stabilized cell surface PVR, which interacted with TIGIT on CD8 + T cells; knockdown of Pvrl1 reduced cell-surface levels of PVR but not levels of Pvr messenger RNA. In Trp53 KO /C-Myc OE mice and mice with tumors grown from Hepa1-6 cells, injection of the combination of anti-PD1 and anti-TIGIT significantly reduced tumor growth, increased the ratio of cytotoxic to regulatory T cells in tumors, and prolonged survival., Conclusions: PVRL1, which is up-regulated by HCC cells, stabilizes cell surface PVR, which interacts with TIGIT, an inhibitory molecule on CD8 + effector memory T cells. This suppresses the ant-tumor immune response. Inhibitors of PVRL1/TIGIT, along with anti-PD1 might be developed for treatment of HCC., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide which lacks effective treatment. Cancer cells experience high levels of oxidative stress due to increased generation of reactive oxygen species (ROS). Increased antioxidant-producing capacity is therefore found in cancer cells to counteract oxidative stress. The thioredoxin system is a ubiquitous mammalian antioxidant system which scavenges ROS, and we demonstrate that it is vital for HCC growth as it maintains intracellular reduction-oxidation (redox) homeostasis. Transcriptome sequencing in human HCC samples revealed significant overexpression of thioredoxin reductase 1 (TXNRD1), the cytosolic subunit and key enzyme of the thioredoxin system, with significant correlations to poorer clinicopathological features and patient survival. Driven by the transcriptional activation of nuclear factor (erythroid-derived 2)-like 2, the master protector against oxidative stress, TXNRD1 counteracts intracellular ROS produced in human HCC. Inhibition of TXNRD1 through genetic inhibition hindered the proliferation of HCC cells and induced apoptosis in vitro. Administration of the pharmacological TXNRD1 inhibitor auranofin (AUR) effectively suppressed the growth of HCC tumors induced using the hydrodynamic tail vein injection and orthotopic implantation models in vivo. Furthermore, AUR sensitized HCC cells toward the conventional therapeutic sorafenib. Conclusion: Our study highlights the reliance of HCC cells on antioxidants for redox homeostasis and growth advantage; targeting TXNRD1 resulted in dramatic accumulation of ROS, which was found to be an effective approach for the suppression of HCC tumor growth., (© 2018 by the American Association for the Study of Liver Diseases.)

Blood vessels in tumors contain chaotic branching structures and leaky vessel lumens, resulting in uneven supply of oxygen in the tumor microenvironment. High metabolic and proliferation rate of tumor cells further depletes the local oxygen supply. Therefore, hypoxia is a common phenomenon in multiple solid malignancies. Hypoxia-inducible factors (HIFs) regulate the transcription of a spectrum of genes, which are vitally important for tumor cell adaption under hypoxia, and shape the tumor microenvironment to become more favorable for progression. HIFs are involved in almost every step of cancer development through inducing angiogenesis, metabolic reprogramming, metastasis, cancer stemness maintenance, chemoresistance, and immune evasion. Here, we describe methods for the assessment of HIF activity, as well as identification of novel transcriptional targets of HIFs in vitro and in vivo.

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications of RNAs have emerged as a new layer of epigenetic regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of eukaryotic messenger RNA (mRNA) and is important for the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that methyltransferase-like 3 (METTL3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors. Clinically, overexpression of METTL3 is associated with poor prognosis of patients with HCC. Functionally, we proved that knockdown of METTL3 drastically reduced HCC cell proliferation, migration, and colony formation in vitro. Knockout of METTL3 remarkably suppressed HCC tumorigenicity and lung metastasis in vivo. On the other hand, using the CRISPR/dCas9-VP64 activation system, we demonstrated that overexpression of METTL3 significantly promoted HCC growth both in vitro and in vivo. Through transcriptome sequencing, m6A sequencing, and m6A methylated RNA immuno-precipitation quantitative reverse-transcription polymerase chain reaction, we identified suppressor of cytokine signaling 2 (SOCS2) as a target of METTL3-mediated m6A modification. Knockdown of METTL3 substantially abolished SOCS2 mRNA m6A modification and augmented SOCS2 mRNA expression. We also showed that m6A-mediated SOCS2 mRNA degradation relied on the m6A reader protein YTHDF2-dependent pathway., Conclusion: METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270)., (© 2017 by the American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O 2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

Background & Aims: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an important epigenetics regulator however, its role in liver carcinogenesis remains to be investigated., Methods: Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing system. Tumor-promoting functions of G9a was studied in both HCC cell lines and nude mice model. The downstream targets of G9a were identified by RNA-Seq and confirmed by ChIP assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo., Results: We identified G9a as a frequently upregulated histone methyltransferase in human HCCs. Upregulation of G9a was significantly associated with HCC progression and aggressive clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and in vivo models. Mechanistically, we showed that the frequent upregulation of G9a in human HCCs was attributed to gene copy number gain at chromosome 6p21. In addition, we identified miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression on G9a and contributed to its upregulation in human HCC. Utilizing RNA sequencing, we identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of RARRES3 contributed to the tumor-promoting function of G9a., Conclusion: This study shows a frequent deregulation of miR-1/G9a/RARRES3 axis in liver carcinogenesis, highlighting the pathological significance of G9a and its therapeutic potential in HCC treatment. Lay summary: In this study, we identified G9a histone methyltransferase was frequently upregulated in human HCC and contributes to epigenetic silencing of tumor suppressor gene RARRES3 in liver cancer. Targeting G9a may be a novel approach for HCC treatment., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Myeloid-derived suppressor cells (MDSCs) possess immunosuppressive activities, which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. Here we report hypoxia as a cause of MDSC accumulation. Using hepatocellular carcinoma (HCC) as a cancer model, we show that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induces ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/CD39L1), in cancer cells, causing its overexpression in HCC clinical specimens. Overexpression of ENTPD2 is found as a poor prognostic indicator for HCC. Mechanistically, we demonstrate that ENTPD2 converts extracellular ATP to 5'-AMP, which prevents the differentiation of MDSCs and therefore promotes the maintenance of MDSCs. We further find that ENTPD2 inhibition is able to mitigate cancer growth and enhance the efficiency and efficacy of immune checkpoint inhibitors. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients, especially those receiving immune therapy.Myeloid-derived suppressor cells (MDSCs) promote tumor immune escape. Here, the authors show that in hepatocellular carcinoma, hypoxia induces the expression of ENTPD2 on cancer cells leading to elevated extracellular 5'-AMP, which in turn promote the maintenance of MDSCs by preventing their differentiation.

Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.

Unlabelled: A population of stromal cells, myeloid-derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia-induced MDSC infiltration is dependent on hypoxia-inducible factors. We further found that hypoxia-inducible factors activate the transcription of chemokine (C-C motif) ligand 26 in cancer cells to recruit chemokine (C-X3-C motif) receptor 1-expressing MDSCs to the primary tumor. Knockdown of chemokine (C-C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C-C motif) ligand 26 production in cancer cells by the hypoxia-inducible factor inhibitor digoxin or blockade of chemokine (C-X3-C motif) receptor 1 in MDSCs by chemokine (C-X3-C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth., Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797-813)., (© 2016 by the American Association for the Study of Liver Diseases.)

Purpose: Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen., Experimental Design: Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan-Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor., Results: NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2., Conclusions: Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105-17. ©2016 AACR., (©2016 American Association for Cancer Research.)

Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH-Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.

Long noncoding RNAs (lncRNAs), generally longer than 200 nucleotides and with poor protein coding potential, are usually considered collectively as a heterogeneous class of RNAs. Recently, an increasing number of studies have shown that lncRNAs can involve in various critical biological processes and a number of complex human diseases. Not only the primary sequences of many lncRNAs are directly interrelated to a specific functional role, strong evidence suggests that their secondary structures are even more interrelated to their known functions. As functional molecules, lncRNAs have become more and more relevant to many researchers. Here, we review recent, state-of-the-art advances in the three levels (the primary sequence, the secondary structure and the function annotation) of the lncRNA research, as well as computational methods for lncRNA data analysis., (© The Author 2015. Published by Oxford University Press.)

There is no effective treatment for intracerebral hemorrhage (ICH). Intracerebral delivery of nanomaterials into the hemorrhagic lesion may be a new therapeutic strategy. In a rat model of ICH plus ultra-early hematoma aspiration, we found that locally delivered self-assembling peptide nanofiber scaffold (SAPNS) replaced the hematoma, reduced acute brain injury and brain cavity formation, and improved sensorimotor functional recovery. SAPNS serves as biocompatible material in the hemorrhagic brain cavity. Local delivery of this nanomaterial may facilitate the repair of ICH related brain injury and functional recovery. From the clinical editor: In a rat model of intracranial hemorrhage, these authors demonstrate that following ultra-early hematoma aspiration, local delivery of a self-assembling peptide nanofiber scaffold replaces the hematoma, reduces brain cavity formation, and improves sensorimotor functional recovery. Similar approaches would be welcome additions to the clinical treatment of this often devastating condition., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.

Unlabelled: Poor prognosis of cancers, including hepatocellular carcinoma (HCC), is mainly associated with metastasis; however, the underlying mechanisms remain poorly understood. This article investigates the role of lysyl oxidase-like 2 (LOXL-2) in the biology of HCC metastasis. First, we showed that HCC metastasis relies on a collagen-modifying enzyme, LOXL2, which was significantly overexpressed in tumorous tissues and sera of HCC patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex, interlinked signaling network that involves multiple regulators, including hypoxia, transforming growth factor beta (TGF-β), and microRNAs (miRNAs), converging to control the expression of LOXL2. We found not only that LOXL2 was regulated by hypoxia/hypoxia-inducible factor 1 alpha (HIF-1α), but also that TGF-β activated LOXL2 transcription through mothers against decapentaplegic homolog 4 (Smad4), whereas two frequently underexpressed miRNA families, miR-26 and miR-29, cooperatively suppressed LOXL2 transcription through interacting with the 3' untranslated region of LOXL2. Third, we demonstrated the imperative roles of LOXL2 in modifying the extracellular matrix components in the tumor microenvironment and metastatic niche of HCC. LOXL2 promoted intrahepatic metastasis by increasing tissue stiffness, thereby enhancing the cytoskeletal reorganization of HCC cells. Furthermore, LOXL2 facilitated extrahepatic metastasis by enhancing recruitment of bone-marrow-derived cells to the metastatic site., Conclusion: These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis., (© 2014 by the American Association for the Study of Liver Diseases.)

The identification of deregulated modules (such as induced by oncogenes) is a crucial step for exploring the pathogenic process of complex diseases. Most of the existing methods focus on deregulation of genes rather than the links of the path among them. In this study, we emphasize on the detection of deregulated links, and develop a novel and effective regulatory path-based approach in finding deregulated modules. Observing that a regulatory pathway between two genes might involve in multiple rather than a single path, we identify condition-specific core regulatory path (CCRP) to detect the significant deregulation of regulatory links. Using time-series gene expression, we define the regulatory strength within each gene pair based on statistical dependence analysis. The CCRPs in regulatory networks can then be identified using the shortest path algorithm. Finally, we derive the deregulated modules by integrating the differential edges (as deregulated links) of the CCRPs between the case and the control group. To demonstrate the effectiveness of our approach, we apply the method to expression data associated with different states of Human Epidermal Growth Factor Receptor 2 (HER2). The experimental results show that the genes as well as the links in the deregulated modules are significantly enriched in multiple KEGG pathways and GO biological processes, most of which can be validated to suffer from impact of this oncogene based on previous studies. Additionally, we find the regulatory mechanism associated with the crucial gene SNAI1 significantly deregulated resulting from the activation of HER2. Hence, our method provides not only a strategy for detecting the deregulated links in regulatory networks, but also a way to identify concerning deregulated modules, thus contributing to the target selection of edgetic drugs.

Background: Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families., Results: The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function., Conclusions: Our results demonstrate that the method we present here using a k-modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

Since cellular functionality is typically envisioned as having a hierarchical structure, we propose a framework to identify modules (or clusters) within protein-protein interaction (PPI) networks in this paper. Based on the within-module and between-module edges of subgraphs and degree distribution, we present a formal module definition in PPI networks. Using the new module definition, an effective quantitative measure is introduced for the evaluation of the partition of PPI networks. Because of the hierarchical nature of functional modules, a hierarchical agglomerative clustering algorithm is developed based on the new measure in order to solve the problem of complexes detection within PPI networks. We use gold standard sets of protein complexes to validate the biological significance of predicted complexes. A comprehensive comparison is performed between our method and other four representative methods. The results show that our algorithm finds more protein complexes with high biological significance and a significant improvement. Furthermore, the predicted complexes by our method, whether dense or sparse, match well with known biological characteristics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Comparative genomics is concerned with the study of genome structure and function of different species. It can provide useful information for the derivation of evolutionary and functional relationships between genomes. Previous work on genome comparison focuses mainly on comparing the entire genomes for visualization without further analysis. As many interesting patterns may exist between genomes and may lead to the discovering of functional gene segments (groups of genes), we propose an algorithm called Multi-Level Genome Comparison Algorithm (MGC) that can be used to facilitate the analysis of genomes at multi-levels during the comparison process to discover sequential and regional consistency in gene segments. Different genomes may have common sub-sequences that differ from each other due to mutations, lateral gene transfers, gene rearrangements, etc., and these sub-sequences are usually not easily identified. Not all the genes can have a perfect one-to-one matching with each other. It is quite possible for one-to-many or many-to-many ambiguous relationships to exist between them. To perform the tasks effectively, MGC takes such ambiguity into consideration during genome comparison by representing genomes in a graph and then make use of a graph mining algorithm called the Multi-Level Attributed Graph Mining Algorithm (MAGMA) to build a hierarchical multi-level graph structure to facilitate genome comparison. To determine the effectiveness of these proposed algorithms, experiments were performed using intra- and inter-species of Microbial genomes. The results show that the proposed algorithms are able to discover multiple level matching patterns that show the similarities and dissimilarities among different genomes, in addition to confirming the specific role of the genes in the genomes.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (> or = 1.2-fold, p<0.05), but only one of them, GDP-dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender-related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.

Porcine liver proteome iTRAQ analysis enabled the confident identification of 880 proteins with a rate of false positive identifications of less than 5%. Proteins involved in energy metabolism, catabolism, protein biosynthesis, electron transport, and other oxidoreductase reactions were highly enriched confirming the central role of liver as the major chemical and energy factory. Comparative analysis with human and mouse liver proteomes demonstrated that 80% of proteins were common to all three liver proteomes. In addition, it was also demonstrated that both sex of the animal and introduction of a novel phytase transgene into the genome each affected around 5% of total liver proteome. After controlling the false discovery rate (FDR

Background: Abel and Trevors have delineated three aspects of sequence complexity, Random Sequence Complexity (RSC), Ordered Sequence Complexity (OSC) and Functional Sequence Complexity (FSC) observed in biosequences such as proteins. In this paper, we provide a method to measure functional sequence complexity., Methods and Results: We have extended Shannon uncertainty by incorporating the data variable with a functionality variable. The resulting measured unit, which we call Functional bit (Fit), is calculated from the sequence data jointly with the defined functionality variable. To demonstrate the relevance to functional bioinformatics, a method to measure functional sequence complexity was developed and applied to 35 protein families. Considerations were made in determining how the measure can be used to correlate functionality when relating to the whole molecule and sub-molecule. In the experiment, we show that when the proposed measure is applied to the aligned protein sequences of ubiquitin, 6 of the 7 highest value sites correlate with the binding domain., Conclusion: For future extensions, measures of functional bioinformatics may provide a means to evaluate potential evolving pathways from effects such as mutations, as well as analyzing the internal structural and functional relationships within the 3-D structure of proteins.

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.

The combined interpretation of gene expression data and gene sequences is important for the investigation of the intricate relationships of gene expression at the transcription level. The expression data produced by microarray hybridization experiments can lead to the identification of clusters of co-expressed genes that are likely co-regulated by the same regulatory mechanisms. By analyzing the promoter regions of co-expressed genes, the common regulatory patterns characterized by transcription factor binding sites can be revealed. Many clustering algorithms have been used to uncover inherent clusters in gene expression data. In this paper, based on experiments using simulated and real data, we show that the performance of these algorithms could be further improved. For the clustering of expression data typically characterized by a lot of noise, we propose to use a two-phase clustering algorithm consisting of an initial clustering phase and a second re-clustering phase. The proposed algorithm has several desirable features: (i) it utilizes both local and global information by computing both a "local" pairwise distance between two gene expression profiles in Phase 1 and a "global" probabilistic measure of interestingness of cluster patterns in Phase 2, (ii) it distinguishes between relevant and irrelevant expression values when performing re-clustering, and (iii) it makes explicit the patterns discovered in each cluster for possible interpretations. Experimental results show that the proposed algorithm can be an effective algorithm for discovering clusters in the presence of very noisy data. The patterns that are discovered in each cluster are found to be meaningful and statistically significant, and cannot otherwise be easily discovered. Based on these discovered patterns, genes co-expressed under the same experimental conditions and range of expression levels have been identified and evaluated. When identifying regulatory patterns at the promoter regions of the co-expressed genes, we also discovered well-known transcription factor binding sites in them. These binding sites can provide explanations for the co-expressed patterns.

Background: In order to understand the intricacy of biomolecules more comprehensively, significant patterns extracted from related data collected from diverse sources must be integrated. These data sources may be local or distributed, possibly with different representation schemes. Often, related data from different sources correspond only with respect to some of their values., Methods: In biological sequence analysis, a goal is to identify new, previously unknown, relevant patterns, to obtain additional insights into the biomolecule. This is known as a pattern discovery task, rather than a pattern matching task. In this research, we present a method to tackle this problem typically found in molecular sequence analysis when the alignment of the sequences is represented as a relation. In this article, we propose an information measure to select attribute values that reflect multiple patterns of significant interdependence information. Based on these selected values, the patterns are evaluated with data values from other sources., Results: In the experiments, a cancer-suppressor gene known as TP53 (encoding tumour protein p53) is analysed with the mutation records of patients. The experiments identify previously unknown points in the molecule that have patterns negatively associated with the occurrence of cancer., Conclusion: Since the evaluated interdependence pattern is a global property of the molecule, we conjecture that the identified points might also be a reflection of the molecule's cancer-suppressor characteristics. The experiments also confirm the usefulness of the proposed method.

Recent advances in mobile technologies have greatly extended traditional communication technologies to mobile devices. At the same time, healthcare environments are by nature "mobile" where doctors and nurses do not have fixed workspaces. Irregular and exceptional events are generated in daily hospital routines, such as operations rescheduling, laboratory/examination results, and adverse drug events. These events may create requests that should be delivered to the appropriate person at the appropriate time. Those requests that are classified as urgent are referred to as alerts. Efficient routing and monitoring of alerts are keys to quality and cost-effective healthcare services. Presently, these are generally handled in an ad hoc manner. In this paper, we propose the use of a healthcare alert management system to handle these alert messages systematically. We develop a model for specifying alerts that are associated with medical tasks and a set of parameters for their routing. We design an alert monitor that matches medical staff and their mobile devices to receive alerts, based on the requirements of these alerts. We also propose a mechanism to handle and reroute, if necessary, an alert message when it has not been acknowledged within a specific deadline.

A pattern analysis approach called event covering is introduced and applied to the problem of determining the significance of measured morphological attributes of cervical cells in an automated screening process. The approach is shown to be effective in deciding which characteristics at the event (outcome) level are of little or no diagnostic value, and in formulating sets of predictive rules using the most pertinent ones.

This article presents a new method for the comparison of multiple macromolecular sequences. It is based on a hierarchical sequence synthesis procedure that does not require any a priori knowledge of the molecular structure of the sequences or the phylogenetic relations among the sequences. It differs from the existing methods as it has the capability of: (i) generating a statistical-structural model of the sequences through a synthesis process that detects homologous groups of the sequences, and (ii) aligning the sequences while the taxonomic tree of the sequences is being constructed in one single phase. It produces superior results when compared with some existing methods.

Multiple sequence comparison refers to the search for similarity in three or more sequences. This article presents a survey of the exhaustive (optimal) and heuristic (possibly sub-optimal) methods developed for the comparison of multiple macromolecular sequences. Emphasis is given to the different approaches of the heuristic methods. Four distance measures derived from information engineering and genetic studies are introduced for the comparison between two alignments of sequences. The use of entropy, which plays a central role in information theory as measures of information, choice and uncertainty, is proposed as a simple measure for the evaluation of the optimality of an alignment in the absence of any a priori knowledge about the structures of the sequences being compared. This article also gives two examples of comparison between alternative alignments of the same set of 5SRNAs as obtained by several different heuristic methods.

Multivariate statistical analysis of a large set of micrographs of biological macromolecules involves the computation of eigenimages representing principal features, on the basis of which similar views of the complexes can be grouped. It is not generally clear what these eigenimages represent physically and which ones should be used in the classification process. In this paper, hierarchical maximum entropy discretisation and event covering are used to (1) detect statistically significant relationships in the eigenimages, (2) select the most relevant eigenimages for classifying biomolecular projections, and (3) build a prototype of the biomolecular complex under study.

This paper presents an unsupervised inference method for determining the higher-order structure from sequence data. The method is general, but in this paper it is applied to nucleic acid sequences in determining the secondary (2-D) and tertiary (3-D) structure of the macromolecule. The method evaluates position - position interdependence of the sequence using an information measure known as expected mutual information. The expected mutual information is calculated for each pair of positions and the chi-square test is used to screen statistically significant position pairs. In the calculation of expected mutual information, an unbiased probability estimator is used to overcome the problem associated with zero observation in conserved sites. A selection criterion based on known structural constraints of the strongest interdependent position pairs is applied yielding position pairs most indicative of secondary and tertiary interactions. The method has been tested using tRNA and 5S rRNA sequences with very good results.

Oncogene expression was examined in the human fetal liver and human hepatomas. Erb (B), erb (A+B), Ha-ras, myc, fos and fms oncogene expression elevated in certain stages of fetal liver development and in hepatoma as compared to the normal adult human liver. In contrast, rel, src, mos, sis, myb, Ki-ras and bas oncogenes showed no apparent change of their mRNA levels during fetal liver development and in hepatoma. Further study of erb B oncogene expression in human cirrhotic liver and hepatoma demonstrated a strong correlation between erb B expression and alteration of its gene structure.

The distribution of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] receptors in isolated rat jejunal cells was investigated in normal and oophorectomized female rats and also in vitamin D-deficient animals. Oophorectomy caused a selective reduction in 1,25-(OH)2D3 receptor numbers in villous cells, the reduction being similar (37%) in both normal and vitamin D-deficient animals. The data suggest that this effect of oophorectomy reflects a direct effect of oestrogens in the intestinal synthesis of 1,25-(OH)2D3 receptors, independent of the renal synthesis of 1,25-(OH)2D3. It is proposed that this phenomenon may, in part, explain the reduction in calcium absorption which occurs after oophorectomy or the menopause.

The distribution of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptors in isolated jejunal villous and crypt cells was investigated in normal and adrenalectomized male rats, and also in animals treated with the synthetic glucocorticoid, dexamethasone, and/or the glucocorticoid antagonist, 11-deoxy-cortisol. Adrenalectomy caused an increase in 1,25-(OH)2D3 receptors whilst dexamethasone treatment led to a reduction in receptor number. 11-Deoxy-cortisol was able to reverse the 'down-regulation' effect caused by glucocorticoids. In all cases, the changes in receptor numbers were more pronounced in crypt cells. The data suggest that, in the small intestine, glucocorticoids may control the synthesis of 1,25-(OH)2D3 receptors via the mediation of a glucocorticoid receptor, and that the adrenal hormones mainly express their effect in crypt cells. It is proposed that this phenomenon may, in part, explain the reduction in calcium absorption which occurs in man after chronic glucocorticoid treatment.

The difficulties in analyzing and clustering (synthesizing) multivariate data of the mixed type (discrete and continuous) are largely due to: 1) nonuniform scaling in different coordinates, 2) the lack of order in nominal data, and 3) the lack of a suitable similarity measure. This paper presents a new approach which bypasses these difficulties and can acquire statistical knowledge from incomplete mixed-mode data. The proposed method adopts an event-covering approach which covers a subset of statistically relevant outcomes in the outcome space of variable-pairs. And once the covered event patterns are acquired, subsequent analysis tasks such as probabilistic inference, cluster analysis, and detection of event patterns for each cluster based on the incomplete probability scheme can be performed. There are four phases in our method: 1) the discretization of the continuous components based on a maximum entropy criterion so that the data can be treated as n-tuples of discrete-valued features; 2) the estimation of the missing values using our newly developed inference procedure; 3) the initial formation of clusters by analyzing the nearest-neighbor distance on subsets of selected samples; and 4) the reclassification of the n-tuples into more reliable clusters based on the detected interdependence relationships. For performance evaluation, experiments have been conducted using both simulated and real life data.