Sporadic renal cell carcinoma in young and elderly patients: are there different clinicopathological features and disease specific survival rates?

BACKGROUND: Sporadic renal cell carcinoma (RCC) is rare in young adults. In the present retrospective study we reviewed clinicopathological features and disease specific survival rates in young patients (≤45 years) with RCC and compared them to old patients (≥75 years) with RCC. METHODS: Between 1992 and 2005 a total of 1042 patients were treated for RCC at our institution. We found 70 patients 45 years or younger (YP) and 150 patients 75 years or older (OP) at time of diagnosis. There were no differences in therapeutical approaches between both groups. Clinical and biologic parameters at diagnosis were compared and subjected to uni- and multivariate analysis to study cancer specific survival and progression rate. Mean postoperative follow-up in both groups was 50.1 months. RESULTS: Mean age was 39 years in YP and 80 years in OP, respectively. YP demonstrated significantly lower stage (pT1-pT2 N0 M0, p = 0.03), lower tumor grade (p = 0.01) and higher male-to-female ratio (p < 0.001). The rate of lymph node metastases or distant metastatic disease at presentation did not differ significantly between both groups. In multivariate analysis young age was independently associated with a higher 5-year cancer specific survival (95.2% vs. 72.3%, p = 0.009) and a lower 5-year progression rate (11.3% vs. 42.5%, p = 0.002). CONCLUSION: Sporadic RCC in young patients have lower tumor stages and grades and a better outcome compared to elderly. Age≤45 years was an independent prognostic factor for survival and progression

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described

Diurnally Entrained Anticipatory Behavior in Archaea

By sensing changes in one or few environmental factors biological systems can anticipate future changes in multiple factors over a wide range of time scales (daily to seasonal). This anticipatory behavior is important to the fitness of diverse species, and in context of the diurnal cycle it is overall typical of eukaryotes and some photoautotrophic bacteria but is yet to be observed in archaea. Here, we report the first observation of light-dark (LD)-entrained diurnal oscillatory transcription in up to 12% of all genes of a halophilic archaeon Halobacterium salinarum NRC-1. Significantly, the diurnally entrained transcription was observed under constant darkness after removal of the LD stimulus (free-running rhythms). The memory of diurnal entrainment was also associated with the synchronization of oxic and anoxic physiologies to the LD cycle. Our results suggest that under nutrient limited conditions halophilic archaea take advantage of the causal influence of sunlight (via temperature) on O2 diffusivity in a closed hypersaline environment to streamline their physiology and operate oxically during nighttime and anoxically during daytime

Pyrosequencing-Based Assessment of Bacterial Community Structure Along Different Management Types in German Forest and Grassland Soils

BACKGROUND: Soil bacteria are important drivers for nearly all biogeochemical cycles in terrestrial ecosystems and participate in most nutrient transformations in soil. In contrast to the importance of soil bacteria for ecosystem functioning, we understand little how different management types affect the soil bacterial community composition. METHODOLOGY/PRINCIPAL FINDINGS: We used pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to identify changes in bacterial diversity and community structure in nine forest and nine grassland soils from the Schwäbische Alb that covered six different management types. The dataset comprised 598,962 sequences that were affiliated to the domain Bacteria. The number of classified sequences per sample ranged from 23,515 to 39,259. Bacterial diversity was more phylum rich in grassland soils than in forest soils. The dominant taxonomic groups across all samples (>1% of all sequences) were Acidobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Firmicutes. Significant variations in relative abundances of bacterial phyla and proteobacterial classes, including Actinobacteria, Firmicutes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes and Alphaproteobacteria, between the land use types forest and grassland were observed. At the genus level, significant differences were also recorded for the dominant genera Phenylobacter, Bacillus, Kribbella, Streptomyces, Agromyces, and Defluviicoccus. In addition, soil bacterial community structure showed significant differences between beech and spruce forest soils. The relative abundances of bacterial groups at different taxonomic levels correlated with soil pH, but little or no relationships to management type and other soil properties were found. CONCLUSIONS/SIGNIFICANCE: Soil bacterial community composition and diversity of the six analyzed management types showed significant differences between the land use types grassland and forest. Furthermore, bacterial community structure was largely driven by tree species and soil pH

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process

Hepatitis C Virus Reveals a Novel Early Control in Acute Immune Response

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response

Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp

[EN] Proanthocyanidins (PAs), or condensed tannins, are powerful antioxidants that remove harmful free oxygen radicals from cells. To engineer the anthocyanin and proanthocyanidin biosynthetic pathways to de novo produce PAs in two Nicotiana species, we incorporated four transgenes to the plant chassis. We opted to perform a simultaneous transformation of the genes linked in a multigenic construct rather than classical breeding or retransformation approaches. We generated a GoldenBraid 2.0 multigenic construct containing two Antirrhinum majus transcription factors (AmRosea1 and AmDelila) to upregulate the anthocyanin pathway in combination with two Medicago truncatula genes (MtLAR and MtANR) to produce the enzymes that will derivate the biosynthetic pathway to PAs production. Transient and stable transformation of Nicotiana benthamiana and Nicotiana tabacum with the multigenic construct were respectively performed. Transient expression experiments in N. benthamiana showed the activation of the anthocyanin pathway producing a purple color in the agroinfiltrated leaves and also the effective production of 208.5 nmol (-) catechin/g FW and 228.5 nmol (-) epicatechin/g FW measured by the p-dimethylaminocinnamaldehyde (DMACA) method. The integration capacity of the four transgenes, their respective expression levels and their heritability in the second generation were analyzed in stably transformed N. tabacum plants. DMACA and phoroglucinolysis/HPLC-MS analyses corroborated the activation of both pathways and the effective production of PAs in T0 and T1 transgenic tobacco plants up to a maximum of 3.48 mg/g DW. The possible biotechnological applications of the GB2.0 multigenic approach in forage legumes to produce "bloatsafe" plants and to improve the efficiency of conversion of plant protein into animal protein (ruminal protein bypass) are discussed.This work was supported by grants BIO2012-39849-C02-01 and BIO2016-75485-R from the Spanish Ministry of Economy and Competitiveness (MINECO) (http://www.idi.mineco.gob.es/portal/site/MICINN) to LAC and a fellowship of the JAE-CSIC program to SF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Fresquet-Corrales, S.; Roque Mesa, EM.; Sarrión-Perdigones, A.; Rochina, M.; López-Gresa, MP.; Díaz-Mula, HM.; Belles Albert, JM.... (2017). Metabolic engineering to simultaneously activate anthocyanin and proanthocyanidin biosynthetic pathways in Nicotiana spp. PLoS ONE. 12(9). https://doi.org/10.1371/journal.pone.0184839Se018483912

Phaeochromocytoma and functioning paraganglioma in childhood and adolescence: role of iodine 131 metaiodobenzylguanidine

Phaeochromocytomas and functioning paragangliomas are rare tumours in childhood and adolescence. We review our experience of 43 cases (24 men, 19 women) who were first diagnosed at the age of ⩽ 18 years. All patients were evaluated at some point in their illness with iodine 131 metaiodobenzylguanidine ( 131 I-mIBG) scintigraphy. Eight patients (19%) had bilateral adrenal tumours, 12 (28%) had solitary extra-adrenal tumours, and 8 (19%) had multiple tumours. In 10 patients (23%), the tumours were associated with a familial neurocristopathic syndrome. Thirteen of 24 (54%) unifocal tumours which were initially considered to be benign ultimately proved to be multi-focal and/or malignant. The final prevalence of malignancy was 60% − 26 patients, of whom only 15 (57%) had obviously malignant tumours at the time of diagnosis. Primary tumour size ⋝5 cm was more commonly associated with a malignant course in adrenal but not extra-adrenal tumours. No other clinical, biochemical or morphological characteristic was significantly associated with malignancy. Although the high prevalence of malignancy in this series at least partly reflects referral bias, the need for lifelong follow-up of these patients is underscored. 131 I-mIBG scintigraphy was positive in 36 patients (84%), with a somewhat lower false-negative rate (12%) than X-ray computed tomography (20%). Eight patients with malignant tumours received therapeutic doses of 131 I-mIBG, with partial tumour responses in 3. Thus, 131 I-mIBG is an efficacious, non-invasive, localising agent and may be considered as a palliative therapeutic agent when alternatives have failed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46831/1/259_2005_Article_BF02262730.pd