In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation

The Waddlia Genome: A Window into Chlamydial Biology

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2′116′312bp chromosome and a 15′593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae

Microsporidia::Why Make Nucleotides if You Can Steal Them?

Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions

Assessment of a novel smartglass-based point-of-care fusion approach for mixed reality-assisted targeted prostate biopsy: A pilot proof-of-concept study

PurposeWhile several biopsy techniques and platforms for magnetic resonance imaging (MRI)-guided targeted biopsy of the prostate have been established, none of them has proven definite superiority. Augmented and virtual reality (mixed reality) smartglasses have emerged as an innovative technology to support image-guidance and optimize accuracy during medical interventions. We aimed to investigate the benefits of smartglasses for MRI-guided mixed reality-assisted cognitive targeted biopsy of the prostate.MethodsFor prospectively collected patients with suspect prostate PIRADS lesions, multiparametric MRI was uploaded to a smartglass (Microsoft® Hololens I), and smartglass-assisted targeted biopsy (SMART TB) of the prostate was executed by generation of a cognitive fusion technology at the point-of-care. Detection rates of prostate cancer (PCA) were compared between SMART TB and 12-core systematic biopsy. Assessment of SMART-TB was executed by the two performing surgeons based on 10 domains on a 10-point scale ranging from bad (1) to excellent (10).ResultsSMART TB and systematic biopsy of the prostate were performed for 10 patients with a total of 17 suspect PIRADS lesions (PIRADS 3, n = 6; PIRADS 4, n = 6; PIRADS 5, n = 5). PCA detection rate per core was significant (p < 0.05) higher for SMART TB (47%) than for systematic biopsy (19%). Likelihood for PCA according to each core of a PIRADS lesion (17%, PIRADS 3; 58%, PIRADS 4; 67%, PIRADS 5) demonstrated convenient accuracy. Feasibility scores for SMART TB were high for practicality (10), multitasking (10), execution speed (9), comfort (8), improvement of surgery (8) and image quality (8), medium for physical stress (6) and device handling (6) and low for device weight (5) and battery autonomy (4).ConclusionSMART TB has the potential to increase accuracy for PCA detection and might enhance cognitive MRI-guided targeted prostate biopsy in the future

Expression of inhibitor of apoptosis protein Livin in renal cell carcinoma and non-tumorous adult kidney

The antiapoptotic Livin/ML-IAP gene has recently gained much attention as a potential new target for cancer therapy. Reports indicating that livin is expressed almost exclusively in tumours, but not in the corresponding normal tissue, suggested that the targeted inhibition of livin may present a novel tumour-specific therapeutic strategy. Here, we compared the expression of livin in renal cell carcinoma and in non-tumorous adult kidney tissue by quantitative real-time reverse transcription-PCR, immunoblotting, and immunohistochemistry. We found that livin expression was significantly increased in tumours (P=0.0077), but was also clearly detectable in non-tumorous adult kidney. Transcripts encoding Livin isoforms α and β were found in both renal cell carcinoma and normal tissue, without obvious qualitative differences. Livin protein in renal cell carcinoma samples exhibited cytoplasmic and/or nuclear staining. In non-tumorous kidney tissue, Livin protein expression was only detectable in specific cell types and restricted to the cytoplasm. Thus, whereas the relative overexpression of livin in renal cell carcinoma indicates that it may still represent a therapeutic target to increase the apoptotic sensitivity of kidney cancer cells, this strategy is likely to be not tumour-specific

Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

Background: Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. Methodology/Principal Findings: In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Conclusions/Significance: Taken together, these data provide a comprehensive molecular characterization of a chloroplas

Fluorescence Lifetime Imaging Unravels C. trachomatis Metabolism and Its Crosstalk with the Host Cell

Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity

A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling

Funding Information: Acknowledgments We thank Dr. Tsuyoshi Nakagawa (Shimane University) for the gift of the gateway vectors, pGWB2, pGWB80, pGWB5, and pGWB3. This work was supported in part by funding from the Program for Promotion of Basic Research Activities for Innovation Bioscience (PROBRAIN) to T.N. and T.A., and CREST, Japan Science and Technology Agency to T.N. and T.A.Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.Peer reviewe

Narrowing the knowledge gaps for melanoma

Cutaneous melanoma originates from pigment producing melanocytes or their precursors and is considered the deadliest form of skin cancer. For the last 40 years, few treatment options were available for patients with late-stage melanoma. However, remarkable advances in the therapy field were made recently, leading to the approval of two new drugs, the mutant BRAF inhibitor vemurafenib and the immunostimulant ipilimumab. Although these drugs prolong patients' lives, neither drug cures the disease completely, emphasizing the need for improvements of current therapies. Our knowledge about the complex genetic and biological mechanisms leading to melanoma development has increased, but there are still gaps in our understanding of the early events of melanocyte transformation and disease progression. In this review, we present a summary of the main contributing factors leading to melanocyte transformation and discuss recent novel findings and technologies that will help answer some of the key biological melanoma questions and lay the groundwork for novel therapies

“Hot standards” for the thermoacidophilic archaeon Sulfolobus solfataricus

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, more recently also for systems biology approaches. Within the Sulfolobus Systems Biology (“SulfoSYS”)-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the “–omics” approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics