Proton irradiation of gold targets for 197(m)Hg production

Introduction Irradiation of gold with protons provides access to no-carrier-added 197mHg and 197Hg. Interests in these radionuclides were awakened by the unique chemical and physical properties of mercury and its compounds combined with convenient nuclear properties like suitable half life (197mHg: T1/2 = 23.8 h, 197Hg: T1/2 = 64.14 h), low energy gamma radiations for imaging, Auger – and conversion electrons for therapy. The high thermal conductivity of gold enables high current irradiations and the monoisotopic natural abundance of 197Au supersedes expensive enrichment of the target material. The 197Au(p,n)197(m)Hg reaction was applied until now only for beam monitoring1, stacked foil meas-urements2 or very small scale tracer production. Material and Methods The irradiations were performed at a Cyclone 18/9 (IBA, Louvain la Neuve, Belgium). Its beam-line was sealed with a 1.0 mm vacuum foil (high purity aluminum, 99.999 %) from Goodfellow (Huntingdon, England). High purity gold disks (23 mm diameter, 2 mm thickness, 99.999% pure, 1 ppm Cu) as target material were purchased from ESPI (Ashland, USA). Gold foils as alternative gold targets (12.5×12.5 mm, 0.25 mm thickness, 99.99+ %, 1 ppm Cu) between an aluminum disk (22 mm diameter, 1 mm thickness, 99.0 %, hard) and an aluminum lid (23 mm diameter, 99.0 %, hard) were purchased from Goodfellow (Huntingdon, England). Hydrochloric acid (30%) and nitric acid (65%) were purchased from Roth (Karlsruhe, Germany) in Rotipuran® Ultra quality. Deionized water with > 18 MΩcm resistivity was prepared by a Milli-Q® system (Millipore, Molsheim, France). For separation of target material and side products a liquid-liquid extraction method (Gold was extracted with methyl isobutyl ketone (MIBK) from 2 M HCl target solution) and an ion exchange method (cation exchange resin (Dowex50W-x8, 100–200 mesh, H+ form) were applied. Results and Conclusion No-carrier-added 197(m)Hg was produced from gold via the 197Au(p,n)197(m)Hg reaction at proton energies of 10 MeV in sufficient quantity and quality for imaging studies. Two different methods were studied for the separation of Hg radionuclides generated from Au targets. The results demonstrate the possibility to produce 197(m)Hg from gold at low proton energies. Combined with the presented radiochemical separation methods, the 197Au(p,n) reaction could be the basis for repeatable production of 197(m)Hg for imaging and therapy research on sufficient activity level

Multi-omic signature of body weight change: results from a population-based cohort study

BACKGROUND: Excess body weight is a major risk factor for cardiometabolic diseases. The complex molecular mechanisms of body weight change-induced metabolic perturbations are not fully understood. Specifically, in-depth molecular characterization of long-term body weight change in the general population is lacking. Here, we pursued a multi-omic approach to comprehensively study metabolic consequences of body weight change during a seven-year follow-up in a large prospective study. METHODS: We used data from the population-based Cooperative Health Research in the Region of Augsburg (KORA) S4/F4 cohort. At follow-up (F4), two-platform serum metabolomics and whole blood gene expression measurements were obtained for 1,631 and 689 participants, respectively. Using weighted correlation network analysis, omics data were clustered into modules of closely connected molecules, followed by the formation of a partial correlation network from the modules. Association of the omics modules with previous annual percentage weight change was then determined using linear models. In addition, we performed pathway enrichment analyses, stability analyses, and assessed the relation of the omics modules with clinical traits. RESULTS: Four metabolite and two gene expression modules were significantly and stably associated with body weight change (P-values ranging from 1.9 × 10−4 to 1.2 × 10−24). The four metabolite modules covered major branches of metabolism, with VLDL, LDL and large HDL subclasses, triglycerides, branched-chain amino acids and markers of energy metabolism among the main representative molecules. One gene expression module suggests a role of weight change in red blood cell development. The other gene expression module largely overlaps with the lipid-leukocyte (LL) module previously reported to interact with serum metabolites, for which we identify additional co-expressed genes. The omics modules were interrelated and showed cross-sectional associations with clinical traits. Moreover, weight gain and weight loss showed largely opposing associations with the omics modules. CONCLUSIONS: Long-term weight change in the general population globally associates with serum metabolite concentrations. An integrated metabolomics and transcriptomics approach improved the understanding of molecular mechanisms underlying the association of weight gain with changes in lipid and amino acid metabolism, insulin sensitivity, mitochondrial function as well as blood cell development and function

Stratification of pancreatic tissue samples for molecular studies: RNA-based cellular annotation procedure

Abstract Background/objectives Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. Methods A five-gene transcriptional signature ( RNA CellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. Results Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. Conclusions Via immediate intra-analytical procedure, our RNA-based stratification RNA CellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information

Diagnosis of Pancreatic Ductal Adenocarcinoma and Chronic Pancreatitis by Measurement of microRNA Abundance in Blood and Tissue

A solid process for diagnosis could have a substantial impact on the successful treatment of pancreatic cancer, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analyzed on microarrays and in part validated by real-time PCR assays. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors. Utilizing the minimally invasive blood test, receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analysis demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis; respective AUC values of 0.973 and 0.950 were obtained. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples with AUC values of 1.0 and 0.937, respectively. In addition, discrimination between cancer and chronic pancreatitis was achieved (AUC = 0.875). Also, several miRNAs were identified that exhibited abundance variations in both tissue and blood samples. The results could have an immediate diagnostic value for the evaluation of tumor reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer

FEZ2 Has Acquired Additional Protein Interaction Partners Relative to FEZ1: Functional and Evolutionary Implications

BACKGROUND: The FEZ (fasciculation and elongation protein zeta) family designation was purposed by Bloom and Horvitz by genetic analysis of C. elegans unc-76. Similar human sequences were identified in the expressed sequence tag database as FEZ1 and FEZ2. The unc-76 function is necessary for normal axon fasciculation and is required for axon-axon interactions. Indeed, the loss of UNC-76 function results in defects in axonal transport. The human FEZ1 protein has been shown to rescue defects caused by unc-76 mutations in nematodes, indicating that both UNC-76 and FEZ1 are evolutionarily conserved in their function. Until today, little is known about FEZ2 protein function. METHODOLOGY/PRINCIPAL FINDINGS: Using the yeast two-hybrid system we demonstrate here conserved evolutionary features among orthologs and non-conserved features between paralogs of the FEZ family of proteins, by comparing the interactome profiles of the C-terminals of human FEZ1, FEZ2 and UNC-76 from C. elegans. Furthermore, we correlate our data with an analysis of the molecular evolution of the FEZ protein family in the animal kingdom. CONCLUSIONS/SIGNIFICANCE: We found that FEZ2 interacted with 59 proteins and that of these only 40 interacted with FEZ1. Of the 40 FEZ1 interacting proteins, 36 (90%), also interacted with UNC-76 and none of the 19 FEZ2 specific proteins interacted with FEZ1 or UNC-76. This together with the duplication of unc-76 gene in the ancestral line of chordates suggests that FEZ2 is in the process of acquiring new additional functions. The results provide also an explanation for the dramatic difference between C. elegans and D. melanogaster unc-76 mutants on one hand, which cause serious defects in the nervous system, and the mouse FEZ1 -/- knockout mice on the other, which show no morphological and no strong behavioural phenotype. Likely, the ubiquitously expressed FEZ2 can completely compensate the lack of neuronal FEZ1, since it can interact with all FEZ1 interacting proteins and additional 19 proteins

MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis

Identification of MicroRNA-21 as a Biomarker for Chemoresistance and Clinical Outcome Following Adjuvant Therapy in Resectable Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The high risk of recurrence following surgical resection provides the rationale for adjuvant therapy. However, only a subset of patients benefit from adjuvant therapy. Identification of molecular markers to predict treatment outcome is therefore warranted. The aim of the present study was to evaluate whether expression of novel candidate biomarkers, including microRNAs, can predict clinical outcome in PDAC patients treated with adjuvant therapy.Formalin-fixed paraffin embedded specimens from a cohort of 82 resected Korean PDAC cases were analyzed for protein expression by immunohistochemistry and for microRNA expression using quantitative Real-Time PCR. Cox proportional hazards model analysis in the subgroup of patients treated with adjuvant therapy (N = 52) showed that lower than median miR-21 expression was associated with a significantly lower hazard ratio (HR) for death (HR = 0.316; 95%CI = 0.166–0.600; P = 0.0004) and recurrence (HR = 0.521; 95%CI = 0.280–0.967; P = 0.04). MiR-21 expression status emerged as the single most predictive biomarker for treatment outcome among all 27 biological and 9 clinicopathological factors evaluated. No significant association was detected in patients not treated with adjuvant therapy. In an independent validation cohort of 45 frozen PDAC tissues from Italian cases, all treated with adjuvant therapy, lower than median miR-21 expression was confirmed to be correlated with longer overall as well as disease-free survival. Furthermore, transfection with anti-miR-21 enhanced the chemosensitivity of PDAC cells.. These data provide evidence that miR-21 may allow stratification for adjuvant therapy, and represents a new potential target for therapy in PDAC

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. METHODOLOGY/PRINCIPAL FINDINGS: We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5' and/or 3' ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. CONCLUSIONS/SIGNIFICANCE: Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental stages allowed us to pinpoint molting stages as hotspots of miRNA expression both in sorts and quantities. Based on the analysis of target genes, we hypothesized that miRNAs regulate development through a particular emphasis on complex stages rather than general regulatory mechanisms

A Draft of the Human Septin Interactome

Background: Septins belong to the GTPase superclass of proteins and have been functionally implicated in cytokinesis and the maintenance of cellular morphology. They are found in all eukaryotes, except in plants. In mammals, 14 septins have been described that can be divided into four groups. It has been shown that mammalian septins can engage in homo- and heterooligomeric assemblies, in the form of filaments, which have as a basic unit a hetero-trimeric core. In addition, it has been speculated that the septin filaments may serve as scaffolds for the recruitment of additional proteins. Methodology/Principal Findings: Here, we performed yeast two-hybrid screens with human septins 1-10, which include representatives of all four septin groups. Among the interactors detected, we found predominantly other septins, confirming the tendency of septins to engage in the formation of homo- and heteropolymeric filaments. Conclusions/Significance: If we take as reference the reported arrangement of the septins 2, 6 and 7 within the heterofilament, (7-6-2-2-6-7), we note that the majority of the observed interactions respect the ""group rule"", i.e. members of the same group (e. g. 6, 8, 10 and 11) can replace each other in the specific position along the heterofilament. Septins of the SEPT6 group preferentially interacted with septins of the SEPT2 group (p<0.001), SEPT3 group (p<0.001) and SEPT7 group (p<0.001). SEPT2 type septins preferentially interacted with septins of the SEPT6 group (p<0.001) aside from being the only septin group which interacted with members of its own group. Finally, septins of the SEPT3 group interacted preferentially with septins of the SEPT7 group (p<0.001). Furthermore, we found non-septin interactors which can be functionally attributed to a variety of different cellular activities, including: ubiquitin/sumoylation cycles, microtubular transport and motor activities, cell division and the cell cycle, cell motility, protein phosphorylation/signaling, endocytosis, and apoptosis.Fundao de Amparo a Pesquisa do Estado Sao Paulo (Fapesp)CAPES: Coordenao de Aperfeioamento de Pessoal de Navel SuperiorConselho Nacional de Pesquisa e Desenvolvimento (CNPq)Laboratorio Nacional de Biociencias-Centro Nacional de Pesquisa em Energia e Materais (LNBio-CNPEM