Search

Although external beam radiotherapy (xRT) is commonly used to treat central nervous system (CNS) tumors in patients of all ages, young children treated with xRT frequently experience life-altering and dose-limiting neurocognitive impairment (NI) while adults do not. The lack of understanding of mechanisms responsible for these differences has impeded the development of neuroprotective treatments. Using a newly developed mouse model of xRT-induced NI, we found that neurocognitive function is impaired by ionizing radiation in a dose- and age-dependent manner, with the youngest animals being most affected. Histologic analysis revealed xRT-driven neuronal degeneration and cell death in neurogenic brain regions in young animals but not adults. BH3 profiling showed that neural stem and progenitor cells, neurons, and astrocytes in young mice are highly primed for apoptosis, rendering them hypersensitive to genotoxic damage. Analysis of single-cell RNA sequencing data revealed that neural cell vulnerability stems from heightened expression of proapoptotic genes including BAX, which is associated with developmental and mitogenic signaling by MYC. xRT induced apoptosis in primed neural cells by triggering a p53- and PUMA-initiated, proapoptotic feedback loop requiring cleavage of BID and culminating in BAX oligomerization and caspase activation. Notably, loss of BAX protected against apoptosis induced by proapoptotic signaling in vitro and prevented xRT-induced apoptosis in neural cells in vivo as well as neurocognitive sequelae. On the basis of these findings, preventing xRT-induced apoptosis specifically in immature neural cells by blocking BAX, BIM, or BID via direct or upstream mechanisms is expected to ameliorate NI in pediatric patients with CNS tumor., Significance: Age- and differentiation-dependent apoptotic priming plays a pivotal role in driving radiotherapy-induced neurocognitive impairment and can be targeted for neuroprotection in pediatric patients., (©2023 The Authors; Published by the American Association for Cancer Research.)

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identify specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Olson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposi's sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu ), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch., Author Summary: Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background & Aims: There is a major unmet need to assess the prognostic impact of antifibrotics in clinical trials because of the slow rate of liver fibrosis progression. We aimed to develop a surrogate biomarker to predict future fibrosis progression., Methods: A fibrosis progression signature (FPS) was defined to predict fibrosis progression within 5 years in patients with hepatitis C virus and nonalcoholic fatty liver disease (NAFLD) with no to minimal fibrosis at baseline (n = 421) and was validated in an independent NAFLD cohort (n = 78). The FPS was used to assess response to 13 candidate antifibrotics in organotypic ex vivo cultures of clinical fibrotic liver tissues (n = 78) and cenicriviroc in patients with nonalcoholic steatohepatitis enrolled in a clinical trial (n = 19, NCT02217475). A serum protein-based surrogate FPS was developed and tested in a cohort of compensated cirrhosis patients (n = 122)., Results: A 20-gene FPS was defined and validated in an independent NAFLD cohort (adjusted odds ratio, 10.93; area under the receiver operating characteristic curve, 0.86). Among computationally inferred fibrosis-driving FPS genes, BCL2 was confirmed as a potential pharmacologic target using clinical liver tissues. Systematic ex vivo evaluation of 13 candidate antifibrotics identified rational combination therapies based on epigallocatechin gallate, which were validated for enhanced antifibrotic effect in ex vivo culture of clinical liver tissues. In patients with nonalcoholic steatohepatitis treated with cenicriviroc, FPS modulation was associated with 1-year fibrosis improvement accompanied by suppression of the E2F pathway. Induction of the PPARα pathway was absent in patients without fibrosis improvement, suggesting a benefit of combining PPARα agonism to improve the antifibrotic efficacy of cenicriviroc. A 7-protein serum protein-based surrogate FPS was associated with the development of decompensation in cirrhosis patients., Conclusion: The FPS predicts long-term fibrosis progression in an etiology-agnostic manner, which can inform antifibrotic drug development., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

This study aims to evaluate the clinical application value of two materials, drug-eluting stent, and biodegradable stent, in the treatment of coronary heart disease. The results show that the therapeutic effects of drug-eluting stents and biodegradable stents are similar. Both treatment methods have high safety and effectiveness. The ideal coronary artery stent should have good biocompatibility, safety, and possibility.

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2 + , CLEC5A high , MARCO low liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention., (© 2021. The Author(s).)

A role of tumor-suppressive activity of p53 in the tumor microenvironment (TME) has been implicated but remains fairly understudied. To address this knowledge gap, we leveraged our MdmxS314A mice as recipients to investigate how implanted tumor cells incapacitate host p53 creating a conducive TME for tumor progression. We found that tumor cell-associated stress induced p53 downregulation in peritumor cells via an MDMX-Ser314 phosphorylation-dependent manner. As a result, an immunosuppressive TME was developed, as reflected by diminished immune cell infiltration into tumors and compromised macrophage M1 polarization. Remarkably, ablation of MDMX-Ser314 phosphorylation attenuated p53 decline in peritumor cells, which was associated with mitigation of immunosuppression and significant tumor growth delay. Our data collectively uncover a novel role of p53 in regulating the tumor immune microenvironment, suggesting that p53 restoration in the TME can be exploited as a potential strategy of anticancer therapy., (© The Author(s) (2020). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

By introduction of trilacunary Keggin-type polyoxometalate to the hydrothermal reaction system of Cr 3+ , Ln 3+ and phthalic acid, a series of novel {Cr 4 Ln 4 } heterometallic clusters with the formula Cs 2 [Cr 4 Ln 4 (μ 4 -O) 4 (μ 3 -O) 4 (C 8 H 4 O 4 ) 4 (H 2 O) 12 ](H 3 SiW 12 O 40 )Cl·23H 2 O (1-Ln, Ln = Ce, Pr, Nd) and [Cr 4 Ln 4 (μ 4 -O) 4 (μ 3 -O) 4 (C 8 H 4 O 4 ) 4 (H 2 O) 10 ](H 6 SiW 12 O 40 )Cl 2 ·18H 2 O (2-Ln, Ln = Sm, Eu, Gd, Tb, Dy, Ho, Er) have been obtained. Single-crystal structural analyses show that 1-Ln and 2-Ln constitute the first cases of Cr-Ln heterometallic clusters stabilized by inorganic polyoxometalate anions and organic ligands. Optical spectra studies demonstrate that 1-Ln and 2-Ln are narrow-gap semiconductors with band gaps of about 1.5 eV. Magnetic investigation shows that compound 2-Dy is a potential single molecule magnet., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

[This corrects the article DOI: 10.1371/journal.pone.0110955.].

Although ΔNp63 is known to promote cancer cell proliferation, the underlying mechanism behind its oncogenic function remains elusive. We report here a functional interplay between ΔNp63 and Δ133p53. These two proteins are co-overexpressed in a subset of human cancers and cooperate to promote cell proliferation. Mechanistically, Δ133p53 binds to ΔNp63 and utilizes its transactivation domain to upregulate GLUT1, GLUT4, and PGM expression driving glycolysis. While increased glycolysis provides cancer cells with anabolic metabolism critical for proliferation and survival, it can be harnessed for selective cancer cell killing. Indeed, we show that tumors overexpressing both ΔNp63 and Δ133p53 exhibit heightened sensitivity to vitamin C that accumulate to a lethal level due to accelerated uptake via overexpressed GLUT1. These observations offer a new therapeutic avenue that could be exploited for clinical applications.

Renewable tissues exhibit heightened sensitivity to DNA damage, which is thought to result from a high level of p53. However, cell proliferation in renewable tissues requires p53 down-regulation, creating an apparent discrepancy between the p53 level and elevated sensitivity to DNA damage. Using a combination of genetic mouse models and pharmacologic inhibitors, we demonstrate that it is p53-regulated MDM2 that functions together with MDMX to regulate DNA damage sensitivity by targeting EZH2 (enhancer of zeste homolog 2) for ubiquitination/degradation. As a methyltransferase, EZH2 promotes H3K27me3, and therefore chromatin compaction, to determine sensitivity to DNA damage. We demonstrate that genetic and pharmacologic interference of the association between MDM2 and MDMX stabilizes EZH2, resulting in protection of renewable tissues from radio-/chemotherapy-induced acute injury. In cells with p53 mutation, there are diminished MDM2 levels, and thus accumulation of EZH2, underpinning the resistant phenotype. Our work uncovers an epigenetic mechanism behind tissue sensitivity to DNA damage, carrying important translation implications., Competing Interests: Conflict of interest statement: Harvard University has filed a provisional patent application on behalf of the investigators claiming some of the concepts contemplated in this publication.

Safety is prerequisite for preventive medicine, but non-toxic agents are generally ineffective as clinical chemoprevention. Here we propose a strategy overcoming this challenge by delivering molecular-targeted agent specifically to the effector cell type to achieve sufficient potency, while circumventing toxicity in the context of cancer chemoprevention. Hepatic myofibroblasts drive progressive fibrosis that results in cirrhosis and liver cancer. In a rat model of cirrhosis-driven liver cancer, a small molecule epidermal growth factor receptor inhibitor, erlotinib, was delivered specifically to myofibroblasts by a versatile nanoparticle-based system, targeting platelet-derived growth factor receptor-beta uniquely expressed on their surface in the liver. With systemic administration of erlotinib, tumor burden was reduced to 31%, which was further improved to 21% by myofibroblast-targeted delivery even with reduced erlotinib dose (7.3-fold reduction with equivalent erlotinib dose) and less hepatocyte damage. These findings demonstrate a strategy, cell type-specific kinase inhibition, for more effective and safer precision cancer chemoprevention., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and S2, determined by our previous transcriptome meta-analysis of multiple clinical hepatocellular carcinoma cohorts, are presented in a panel of hepatoma cell lines widely used by the research community. Interestingly, cell line that resembles gene expression pattern of S3 subtype, representing less aggressive tumors, was not identified in the panel. MYC pathway-activated S2-like cell lines showed higher sensitivity to a small molecule BET bromodomain inhibitor, (+)-JQ1, which has anti-MYC activity. These results support the use of hepatoma cell lines as models to evaluate molecular subtype-specific drug response, which is expected to lead to development of tailored, precision care of the patients with hepatocellular carcinoma.

Krüppel-like factor 6 (KLF6) is a transcription factor and tumor suppressor. We previously identified KLF6 as mediator of hepatocyte glucose and lipid homeostasis. The loss or reduction of KLF6 is linked to the progression of hepatocellular carcinoma, but its contribution to liver regeneration and repair in acute liver injury are lacking so far. Here we explore the role of KLF6 in acute liver injury models in mice, and in patients with acute liver failure (ALF). KLF6 was induced in hepatocytes in ALF, and in both acetaminophen (APAP)- and carbon tetrachloride (CCl 4 )-treated mice. In mice with hepatocyte-specific Klf6 knockout (DeltaKlf6), cell proliferation following partial hepatectomy (PHx) was increased compared to controls. Interestingly, key autophagic markers and mediators LC3-II, Atg7 and Beclin1 were reduced in DeltaKlf6 mice livers. Using luciferase assay and ChIP, KLF6 was established as a direct transcriptional activator of ATG7 and BECLIN1, but was dependent on the presence of p53. Here we show, that KLF6 expression is induced in ALF and in the regenerating liver, where it activates autophagy by transcriptional induction of ATG7 and BECLIN1 in a p53-dependent manner. These findings couple the activity of an important growth inhibitor in liver to the induction of autophagy in hepatocytes.

Objective: Advanced hepatocellular carcinoma (HCC) is a lethal malignancy with limited treatment options. Palbociclib, a well-tolerated and selective CDK4/6 inhibitor, has shown promising results in the treatment of retinoblastoma (RB1)-positive breast cancer. RB1 is rarely mutated in HCC, suggesting that palbociclib could potentially be used for HCC therapy. Here, we provide a comprehensive characterisation of the efficacy of palbociclib in multiple preclinical models of HCC., Design: The effects of palbociclib on cell proliferation, cellular senescence and cell death were investigated in a panel of human liver cancer cell lines, in ex vivo human HCC samples, in a genetically engineered mouse model of liver cancer, and in human HCC xenografts in vivo. The mechanisms of intrinsic and acquired resistance to palbociclib were assessed in human liver cancer cell lines and human HCC samples by protein and gene expression analyses., Results: Palbociclib suppressed cell proliferation in human liver cancer cell lines by promoting a reversible cell cycle arrest. Intrinsic and acquired resistance to palbociclib was determined by loss of RB1. A signature of 'RB1 loss of function' was found in <30% of HCC samples. Palbociclib, alone or combined with sorafenib, the standard of care for HCC, impaired tumour growth in vivo and significantly increased survival., Conclusions: Palbociclib shows encouraging results in preclinical models of HCC and represents a novel therapeutic strategy for HCC treatment, alone or particularly in combination with sorafenib. Palbociclib could potentially benefit patients with RB1-proficient tumours, which account for 70% of all patients with HCC., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Purpose of Review: Current clinical practice guidelines recommend regular hepatocellular carcinoma (HCC) surveillance with biannual ultrasound with or without serum alpha-fetoprotein uniformly applied to all patients with cirrhosis. However, clinical implementation of this one-size-fits-all strategy has been challenging as evidenced by very low application rate below 20% due to various reasons, including suboptimal performance of the surveillance modalities., Recent Findings: Newly emerging imaging techniques such as abbreviated MRI (AMRI) and molecular HCC risk biomarkers have increasingly become available for clinical evaluation and implementation. These technologies may have a potential to reshape HCC surveillance by enabling tailored strategies. This would involve performing optimized surveillance tests according to individual HCC risk, and allocating limited medical resources for HCC surveillance based on cost-effectiveness., Summary: Tailored HCC surveillance could lead to achievement of precision HCC care and substantial improvement of the current dismal patient prognosis., Competing Interests: Conflict of Interest Nicolas Goossens, C. Billie Bian, and Yujin Hoshida each declare no potential conflicts of interest.

Cirrhosis is a milieu that develops hepatocellular carcinoma (HCC), the second most lethal cancer worldwide. HCC prediction and prevention in cirrhosis are key unmet medical needs. Here we have established an HCC risk gene signature applicable to all major HCC etiologies: hepatitis B/C, alcohol, and non-alcoholic steatohepatitis. A transcriptome meta-analysis of >500 human cirrhotics revealed global regulatory gene modules driving HCC risk and the lysophosphatidic acid pathway as a central chemoprevention target. Pharmacological inhibition of the pathway in vivo reduced tumors and reversed the gene signature, which was verified in organotypic ex vivo culture of patient-derived fibrotic liver tissues. These results demonstrate the utility of clinical organ transcriptome to enable a strategy, namely, reverse-engineering precision cancer prevention., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nicotinic acid (NA) acting as the precursor of NAD(+)/NADH and NADP(+)/NADPH, participates in many biochemical processes, e.g. lipid metabolism. The main purpose of this study was to investigate the effects of dietary NA on carcass traits, meat quality, blood metabolites, and fat deposition in Chinese crossbred finishing steers. Sixteen steers with the similar body weight and at the age of 24 months were randomly allocated into control group (feeding basal diet) and NA group (feeding basal diet + 1000 mg/kg NA). All experimental cattle were fed a 90% concentrate diet and 10% forage straw in a 120-day feeding experiment. The results showed that supplemental NA in diet increased longissimus area, intramuscular fat content (17.14% vs. 9.03%), marbling score (8.08 vs. 4.30), redness (a*), and chroma (C*) values of LD muscle, but reduced carcass fat content (not including imtramuscular fat), pH24 h and moisture content of LD muscle, along with no effect on backfat thickness. Besides, NA supplementation increased serum HDL-C concentration, but decreased the serum levels of LDL-C, triglyceride, non-esterified fatty acid, total cholesterol, and glycated serum protein. In addition, NA supplementation increased G6PDH and ICDH activities of LD muscle. These results suggested that NA supplementation in diet improves the carcass characteristics and beef quality, and regulates the compositions of serum metabolites. Based on the above results, NA should be used as the feed additive in cattle industry., (© 2016 by the Society for Experimental Biology and Medicine.)

The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.)

TRAF6 plays a crucial role in the regulation of the innate and adaptive immune responses. Although studies have shown that TRAF6 has oncogenic activity, the role of TRAF6 in melanoma is unclear. Here, we report that TRAF6 is overexpressed in primary as well as metastatic melanoma tumors and melanoma cell lines. Knockdown of TRAF6 with shRNA significantly suppressed malignant phenotypes including cell proliferation, anchorage-independent cell growth and metastasis in vitro and in vivo. Notably, we demonstrated that Basigin (BSG)/CD147, a critical molecule for cancer cell invasion and metastasis, is a novel interacting partner of TRAF6. Furthermore, depletion of TRAF6 by shRNA reduced the recruitment of BSG to the plasma membrane and K63-linked ubiquitination, in turn, which impaired BSG-dependent MMP9 induction. Taken together, our findings indicate that TRAF6 is involved in regulating melanoma invasion and metastasis, suggesting that TRAF6 may be a potential target for therapy or chemo-prevention in melanoma.

Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

An experiment was conducted to determine the effects of soy isoflavone daidzein on carcass characteristics, fat deposition, meat quality, and blood metabolites in finishing steers. Fourteen crossbred steers were used in a 120-d finishing study. These steers were stratified by weight into groups and randomly allotted by group to one of two dietary treatments: (1) control and (2) daidzein (500 mg/kg concentrate). The steers were fed a 90% concentrate diet. Supplemental daidzein did not affect slaughter weight, hot carcass weight, and dressing percentage, but tended to reduce fat proportion (not including intramuscular fat) in carcass and backfat thickness of steers. The carcass bone proportion was greater in steers fed daidzein diets than those fed control diets. Daidzein supplementation reduced pH at 24 h after slaughtered and moisture content and increased isocitrate dehydrogenase activity, fat content (16.28% and 7.94%), marbling score (5.29 and 3.36), redness (a*), and chroma (C*) values in longissimus muscle relative to control treatment. The concentrations of blood metabolites including glucose, blood urea nitrogen, triglyceride, total cholesterol, non-esterified fatty acid, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were all lower in steers fed daidzein diets than those fed control diets. Current results suggest that supplemental daidzein can affect lipid metabolism, increase intramuscular fat content and marbling score, and improve meat quality in finishing steers. Daidzein should be a promising feed additive for production of high-quality beef meat., (© 2014 by the Society for Experimental Biology and Medicine.)

Mesothelial cells are fundamental to the maintenance of serosal integrity and homeostasis and play a critical role in normal serosal repair following injury. However, when normal repair mechanisms breakdown, mesothelial cells take on a profibrotic role, secreting inflammatory, and profibrotic mediators, differentiating and migrating into the injured tissues where they contribute to fibrogenesis. The development of new molecular and cell tracking techniques has made it possible to examine the origin of fibrotic cells within damaged tissues and to elucidate the roles they play in inflammation and fibrosis. In addition to secreting proinflammatory mediators and contributing to both coagulation and fibrinolysis, mesothelial cells undergo mesothelial-to-mesenchymal transition, a process analogous to epithelial-to-mesenchymal transition, and become fibrogenic cells. Fibrogenic mesothelial cells have now been identified in tissues where they have not previously been thought to occur, such as within the parenchyma of the fibrotic lung. These findings show a direct role for mesothelial cells in fibrogenesis and open therapeutic strategies to prevent or reverse the fibrotic process.

Gli transcription factors of the Hedgehog (Hh) pathway have been reported to be drivers of malignant mesothelioma (MMe) cell survival. The Gli inhibitor GANT61 induces apoptosis in various cancer cell models, and has been associated directly with Gli inhibition. However various chemotherapeutics can induce cell death through generation of reactive oxygen species (ROS) but whether ROS mediates GANT61-induced apoptosis is unknown. In this study human MMe cells were treated with GANT61 and the mechanisms regulating cell death investigated. Exposure of MMe cells to GANT61 led to G1 phase arrest and apoptosis, which involved ROS but not its purported targets, GLI1 or GLI2. GANT61 triggered ROS generation and quenching of ROS protected MMe cells from GANT61-induced apoptosis. Furthermore, we demonstrated that mitochondria are important in mediating GANT61 effects: (1) ROS production and apoptosis were blocked by mitochondrial inhibitor rotenone; (2) GANT61 promoted superoxide formation in mitochondria; and (3) mitochondrial DNA-deficient LO68 cells failed to induce superoxide, and were more resistant to apoptosis induced by GANT61 than wild-type cells. Our data demonstrate for the first time that GANT61 induces apoptosis by promoting mitochondrial superoxide generation independent of Gli inhibition, and highlights the therapeutic potential of mitochondrial ROS-mediated anticancer drugs in MMe.

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule--4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK-mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3. Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.

A pot experiment was conducted to study the influences of different NaCl concentrations (0, 50, 100, 200, 400 mmol x L(-1)) on the growth, leaf hydrogen peroxide (H2O2), malonaldehyde (MDA) content, superoxide dismutases (SOD), catalases (CAT), peroxidase (POD), ascorbate peroxidase (APX) enzymes activity, water potential, soluble sugar and proline contents of Nitraria roborowskii. The results showed that the growth of N. roborowskii was not affected at lower NaCl concentrations (< or = 50 mmol L(-1)), while the SOD, POD, CAT and APX activities in leaves of N. roborowskii were increased. However, higher NaCl concentrations (> 50 mmol x L(-1)) restrained the growth parameters of crown area, number of branches, dry mass of leaf, branch and lateral root, and remarkably reduced the SOD, POD and CAT activities, soluble sugar and proline contents in leaves of N. roborowskii. H2O2 and MDA contents in leaves were increased and water potential was reduced with increasing NaCl concentrations.

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

Background: The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown., Methodology/principal Findings: Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69&#95;70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour., Conclusions/significance: We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.

Background: Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin's multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained., Methods: To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers., Results: We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death., Conclusions: Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.

The safety, stability, and ability for repeat homologous vaccination makes the DNA vaccine platform an excellent candidate for an effective HIV-1 vaccine. However, the immunogenicity of early DNA vaccines did not translate from small animal models into larger non-human primates and was markedly lower than viral vectors. In addition to improvements to the DNA vector itself, delivery with electroporation, the inclusion of molecular adjuvants, and heterologous prime-boost strategies have dramatically improved the immunogenicity of DNA vaccines for HIV and currently makes them a leading platform with many areas warranting further research and clinical development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

An essential oil extract, derived from the rhizome of Curcuma wenyujin (CWE), possesses antioxidative, antimicrobial, and anti-inflammatory properties. However, it remains unknown how exactly CWE inhibits tumor growth. In this study, using human cervical cancer HeLa cells, the authors postulated that CWE has the ability to inhibit tumor growth. The study shows that CWE dose-dependently suppressed colony formation and inhibited the proliferation of HeLa cells through blockade of cell cycle progression at G1 phase and apoptosis. CWE-induced G1 arrest was associated with retinoblastoma protein dephosphorylation and reduced amounts of cyclins D1 and D3, and cyclin-dependent kinase 4 and 6 proteins. CWE treatment resulted in apoptosis in HeLa cells as evidenced by morphological changes, caspase activation and PARP cleavage, which can be reversed by a pan-caspase inhibitor. It was observed that CWE treatment activated the mitochondrial apoptotic pathway indicated by a decrease in Mcl-1 and Bcl-xL levels, resulting in mitochondrial membrane potential loss and caspases 9 activation. CWE-treated cells displayed reduced PTEN, AKT, and STAT3 phosphorylation and downregulation of NFkappaB signaling, providing a mechanism for the G1 arrest and apoptosis observed. Furthermore, CWE inhibited tumor growth of HeLa in a xenograft mouse tumor model, suggesting that CWE inhibited tumorigenesis by inhibiting cell proliferation and inducing apoptosis. These findings are the first to reveal the molecular basis for the anticervical cancer action of CWE. The results suggest that CWE could be developed as a drug for the management of cervical cancer.

Kruppel-like factor 4 (KLF4) belongs to a family of evolutionarily conserved zinc finger-containing transcription factors. It has been shown to mediate self renewal and pluripotency, regulate adipogenesis and play a critical role in monocyte differentiation. KLF4 is also highly expressed in squamous cell carcinomas and in 70% of all primary human breast cancers, suggesting a putative role for KLF4 as being an oncogene and as an antiapoptotic factor. However, the mechanism of this regulation remains unclear. Here, we show that KLF4 is induced during histone deacetylase inhibitor treatment, and regulates the extrinsic apoptosis pathway by inhibiting caspase cleavage. In addition, KLF4 binds to the p57(Kip2) promoter and transcriptionally upregulates its expression, which in turn inhibits the stress activated protein kinase cascade and c-Jun phosphorylation. Our findings indicate that in cancer cells that express high levels of KLF4 may be refractory to HDACi treatment. Results of our study demonstrate an unexpected antiapoptotic function of KLF4, and suggest an important cell fate determinant following histone deacetylase inhibitor induced apoptosis.

Background: FAT10 is a member of the ubiquitin-like-modifier family of proteins. Over-expression of the FAT10 gene was observed in the tumors of several epithelial cancers. High FAT10 expression was found to lead to increased chromosome instability via the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell-cycle. FAT10 expression was also previously reported to be regulated by cytokines and p53., Results: Here, we report that FAT10 expression is regulated at the protein and transcript level during cell-cycle with highest expression observed during the S-phase of the cell-cycle. The distal region between -1997 to -975 bp from the transcription start site of the FAT10 promoter may play a role in the repression of FAT10 expression during G2/M phase of the cell-cycle., Conclusion: FAT10 expression is regulated during cell-cycle.

Nosocomial isolates of Pseudomonas aeruginosa exhibit high rates of resistance to antibiotics, and are often multidrug resistant. P. aeruginosa clinical isolates (n = 56) were obtained from ICU patients in a hospital in Pakistan over a 3-y period. Antimicrobial susceptibility of the 56 P. aeruginosa clinical isolates was investigated using 7 antibiotics and the resistance rates were as follows: aztreonam (68% resistant), ceftazidime (67%), imipenem (66%), ofloxacin (59%), amikacin (56%), gentamicin (44%), and piperacillin-tazobactam (27%) (p < 0.01). In addition, 55% of the P. aeruginosa clinical isolates were resistant to 4 or more antibiotics. Imipenem-resistant strains were frequently associated with ceftazidime, ofloxacin, aztreonam, and more strikingly, amikacin resistance (p < 0.05). PCR (using P. aeruginosa-specific primers VIC1 + VIC2 and P1 + P2, respectively) was highly specific and sensitive, and was positive for all 56 P. aeruginosa isolates tested. Automated ribotyping was used to investigate the clonal diversity of the 56 P. aeruginosa isolates. Automated ribotyping indicated that the clinical isolates were clonally related and could be clustered into 4 major ribogroups based on their similarity index, with ribogroup II being the dominant one. The P. aeruginosa isolates in ribogroup II were correlated with their antibiotic resistance pattern and, interestingly, there seemed to be a gradual acquisition of multiple antibiotic resistance associated with the isolates within this group over time. The ribotyping data, together with the antibiotic resistance profile, provide valuable molecular epidemiology information for the control of hospital-acquired P. aeruginosa infections.