Degradation of HIF-1alpha under Hypoxia Combined with Induction of Hsp90 Polyubiquitination in Cancer Cells by Hypericin: a Unique Cancer Therapy

The perihydroxylated perylene quinone hypericin has been reported to possess potent anti-metastatic and antiangiogenic activities, generated by targeting diverse crossroads of cancer-promoting processes via unique mechanisms. Hypericin is the only known exogenous reagent that can induce forced poly-ubiquitination and accelerated degradation of heat shock protein 90 (Hsp90) in cancer cells. Hsp90 client proteins are thereby destabilized and rapidly degraded. Hsp70 client proteins may potentially be also affected via preventing formation of hsp90-hsp70 intermediate complexes. We show here that hypericin also induces enhanced degradation of hypoxia-inducible factor 1α (HIF-1α) in two human tumor cell lines, U87-MG glioblastoma and RCC-C2VHL−/− renal cell carcinoma and in the non-malignant ARPE19 retinal pigment epithelial cell line. The hypericin-accelerated turnover of HIF-1α, the regulatory precursor of the HIF-1 transcription factor which promotes hypoxic stress and angiogenic responses, overcomes the physiologic HIF-1α protein stabilization which occurs in hypoxic cells. The hypericin effect also eliminates the high HIF-1α levels expressed constitutively in the von-Hippel Lindau protein (pVHL)-deficient RCC-C2VHL−/− renal cell carcinoma cell line. Unlike the normal ubiquitin-proteasome pathway-dependent turnover of HIF-α proteins which occurs in normoxia, the hypericin-induced HIF-1α catabolism can occur independently of cellular oxygen levels or pVHL-promoted ubiquitin ligation of HIF-1α. It is mediated by lysosomal cathepsin-B enzymes with cathepsin-B activity being optimized in the cells through hypericin-mediated reduction in intracellular pH. Our findings suggest that hypericin may potentially be useful in preventing growth of tumors in which HIF-1α plays pivotal roles, and in pVHL ablated tumor cells such as renal cell carcinoma through elimination of elevated HIF-1α contents in these cells, scaling down the excessive angiogenesis which characterizes these tumors

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT

Iron homeostasis and oxidative stress in idiopathic pulmonary alveolar proteinosis: a case-control study

<p>Abstract</p> <p>Background</p> <p>Lung injury caused by both inhaled dusts and infectious agents depends on increased availability of iron and metal-catalyzed oxidative stress. Because inhaled particles, such as silica, and certain infections can cause secondary pulmonary alveolar proteinosis (PAP), we tested the hypothesis that idiopathic PAP is associated with an altered iron homeostasis in the human lung.</p> <p>Methods</p> <p>Healthy volunteers (n = 20) and patients with idiopathic PAP (n = 20) underwent bronchoalveolar lavage and measurements were made of total protein, iron, tranferrin, transferrin receptor, lactoferrin, and ferritin. Histochemical staining for iron and ferritin was done in the cell pellets from control subjects and PAP patients, and in lung specimens of patients without cardiopulmonary disease and with PAP. Lavage concentrations of urate, glutathione, and ascorbate were also measured as indices of oxidative stress.</p> <p>Results</p> <p>Lavage concentrations of iron, transferrin, transferrin receptor, lactoferrin, and ferritin were significantly elevated in PAP patients relative to healthy volunteers. The cells of PAP patients had accumulated significant iron and ferritin, as well as considerable amounts of extracellular ferritin. Immunohistochemistry for ferritin in lung tissue revealed comparable amounts of this metal-storage protein in the lower respiratory tract of PAP patients both intracellularly and extracellularly. Lavage concentrations of ascorbate, glutathione, and urate were significantly lower in the lavage fluid of the PAP patients.</p> <p>Conclusion</p> <p>Iron homeostasis is altered in the lungs of patients with idiopathic PAP, as large amounts of catalytically-active iron and low molecular weight anti-oxidant depletion are present. These findings suggest a metal-catalyzed oxidative stress in the maintenance of this disease.</p

Health Newscasts for Increasing Influenza Vaccination Coverage: An Inductive Reasoning Game Approach

Both pandemic and seasonal influenza are receiving more attention from mass media than ever before. Topics such as epidemic severity and vaccination are changing the way in which we perceive the utility of disease prevention. Voluntary influenza vaccination has been recently modeled using inductive reasoning games. It has thus been found that severe epidemics may occur because individuals do not vaccinate and, instead, attempt to benefit from the immunity of their peers. Such epidemics could be prevented by voluntary vaccination if incentives were offered. However, a key assumption has been that individuals make vaccination decisions based on whether there was an epidemic each influenza season; no other epidemiological information is available to them. In this work, we relax this assumption and investigate the consequences of making more informed vaccination decisions while no incentives are offered. We obtain three major results. First, individuals will not cooperate enough to constantly prevent influenza epidemics through voluntary vaccination no matter how much they learned about influenza epidemiology. Second, broadcasting epidemiological information richer than whether an epidemic occurred may stabilize the vaccination coverage and suppress severe influenza epidemics. Third, the stable vaccination coverage follows the trend of the perceived benefit of vaccination. However, increasing the amount of epidemiological information released to the public may either increase or decrease the perceived benefit of vaccination. We discuss three scenarios where individuals know, in addition to whether there was an epidemic, (i) the incidence, (ii) the vaccination coverage and (iii) both the incidence and the vaccination coverage, every influenza season. We show that broadcasting both the incidence and the vaccination coverage could yield either better or worse vaccination coverage than broadcasting each piece of information on its own

A Membrane-Bound Vertebrate Globin

The family of vertebrate globins includes hemoglobin, myoglobin, and other O2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O2 binding with a variable affinity (P50∼1.3–12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein

Identification of errors introduced during high throughput sequencing of the T cell receptor repertoire

<p>Abstract</p> <p>Background</p> <p>Recent advances in massively parallel sequencing have increased the depth at which T cell receptor (TCR) repertoires can be probed by >3log10, allowing for saturation sequencing of immune repertoires. The resolution of this sequencing is dependent on its accuracy, and direct assessments of the errors formed during high throughput repertoire analyses are limited.</p> <p>Results</p> <p>We analyzed 3 monoclonal TCR from TCR transgenic, Rag^-/-mice using Illumina^®sequencing. A total of 27 sequencing reactions were performed for each TCR using a trifurcating design in which samples were divided into 3 at significant processing junctures. More than 20 million complementarity determining region (CDR) 3 sequences were analyzed. Filtering for lower quality sequences diminished but did not eliminate sequence errors, which occurred within 1-6% of sequences. Erroneous sequences were pre-dominantly of correct length and contained single nucleotide substitutions. Rates of specific substitutions varied dramatically in a position-dependent manner. Four substitutions, all purine-pyrimidine transversions, predominated. Solid phase amplification and sequencing rather than liquid sample amplification and preparation appeared to be the primary sources of error. Analysis of polyclonal repertoires demonstrated the impact of error accumulation on data parameters.</p> <p>Conclusions</p> <p>Caution is needed in interpreting repertoire data due to potential contamination with mis-sequence reads. However, a high association of errors with phred score, high relatedness of erroneous sequences with the parental sequence, dominance of specific nt substitutions, and skewed ratio of forward to reverse reads among erroneous sequences indicate approaches to filter erroneous sequences from repertoire data sets.</p

The integrated analysis of metabolic and protein interaction networks reveals novel molecular organizing principles

Background: The study of biological interaction networks is a central theme of systems biology. Here, we investigate the relationships between two distinct types of interaction networks: the metabolic pathway map and the protein-protein interaction network (PIN). It has long been established that successive enzymatic steps are often catalyzed by physically interacting proteins forming permanent or transient multi-enzymes complexes. Inspecting high-throughput PIN data, it was shown recently that, indeed, enzymes involved in successive reactions are generally more likely to interact than other protein pairs. In our study, we expanded this line of research to include comparisons of the underlying respective network topologies as well as to investigate whether the spatial organization of enzyme interactions correlates with metabolic efficiency. Results: Analyzing yeast data, we detected long-range correlations between shortest paths between proteins in both network types suggesting a mutual correspondence of both network architectures. We discovered that the organizing principles of physical interactions between metabolic enzymes differ from the general PIN of all proteins. While physical interactions between proteins are generally dissortative, enzyme interactions were observed to be assortative. Thus, enzymes frequently interact with other enzymes of similar rather than different degree. Enzymes carrying high flux loads are more likely to physically interact than enzymes with lower metabolic throughput. In particular, enzymes associated with catabolic pathways as well as enzymes involved in the biosynthesis of complex molecules were found to exhibit high degrees of physical clustering. Single proteins were identified that connect major components of the cellular metabolism and may thus be essential for the structural integrity of several biosynthetic systems. Conclusion: Our results reveal topological equivalences between the protein interaction network and the metabolic pathway network. Evolved protein interactions may contribute significantly towards increasing the efficiency of metabolic processes by permitting higher metabolic fluxes. Thus, our results shed further light on the unifying principles shaping the evolution of both the functional (metabolic) as well as the physical interaction network

Reproductive Schedules in Southern Bluefin Tuna: Are Current Assumptions Appropriate?

Southern bluefin tuna (SBT) appear to comprise a single stock that is assumed to be both mixed across its distribution and having reproductive adults that are obligate, annual spawners. The putative annual migration cycle of mature SBT consists of dispersed foraging at temperate latitudes with migration to a single spawning ground in the tropical eastern Indian Ocean. Spawning migrations have been assumed to target two peaks in spawning activity; one in September-October and a second in February-March. SBT of sizes comparable to that of individuals observed on the spawning ground were satellite tagged in the Tasman Sea region (2003–2008) and demonstrated both migrations to the spawning grounds and residency in the Tasman Sea region throughout the whole year. All individuals undertaking apparent spawning migrations timed their movements to coincide with the second recognised spawning peak or even later. These observations suggest that SBT may demonstrate substantial flexibility in the scheduling of reproductive events and may even not spawn annually as currently assumed. Further, the population on the spawning grounds may be temporally structured in association with foraging regions. These findings provide new perspectives on bluefin population and spatial dynamics and warrant further investigation and consideration of reproductive schedules in this species