In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral  Here we2.A large number of genes have been tested for their therapeutic efficacy against pancreatic tumors. The re-introduction of genes capable to activate cell death in tumoral cells or genes that can modulate intrinsic cellular factors and eliminate cancer cells are among the most common approaches. As cancer cells present many up-regulated genes or express mutated genes, silencing or gene-knockdown of undesired genes have also been studied.2.1.Apoptosis resistance seems to be a hallmark of cancer that largely accounts for the aggressive nature of pancreatic cancer. The re-engagement of the disrupted apoptosis program in pancreatic cancer offers a compelling general strategy for effective and tumor-cell-specific cancer therapy. Studies targeting single apoptosis-associated genes deregulated in pancreatic cancer have shown encouraging results. For example, downregulation of the anti-apoptotic gene BCL-2 triggered anti-proliferative and pro-apoptotic effects in tumor cells but not in non-malignant tissues . Interes2.2.The inhibition of oncogene expression and restoration of tumor suppressor functions have been studied in pancreatic cancer gene therapy. The most common approaches have targeted key mutated genes in pancreatic neoplasia. Strategies involving the silencing of mutated K-ras and/or the functional rescue of p53 or p16 tumor suppressor genes have Shown significant antitumoral responses in mouse models . Due to 2.3.Suicide or prodrug-converting cancer gene therapy is based on the transfer of an enzyme able to transform a prodrug into a toxic metabolite, resulting in cell death. The herpes simplex virus thymidine kinase (TK) gene in combination with the prodrug ganciclovir (GCV) is the most widely described suicide gene therapy. It consists of the transfer of the TK gene to the tumoral cells, followed by the administration of the nucleoside analogue GCV. TK enzyme converts GCV to its monophosphate form, that is later converted by cellular enzymes into GCV triphosphate and is incorporated into the genome, triggering the formation of double-strand breaks and, finally, causing cell death by apoptosis ,11. The in vivo  and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which aids the spread of the activated prodrug through the tumor, increased tumor shrinkage by five- to 10-fold in vivo compared to the non-armed virus [HSV-1 oncolytic viruses have been studied to treat pancreatic cancer. Intraperitoneal delivery of the replication-conditional hrR3 (a mutant containing an insertion of the lac-Z gene in the ribonucleotide reductase gene) followed by GCV, improved survival in a murine model of disseminated pancreatic cancer . In anoted virus .Oncolytic armed vaccinia viruses have also been generated to act against pancreatic cancer. Vaccinia has a natural oncolytic capacity due to the interference of replication in normal cells by the interferon, while the low interferon production in tumoral cells allows virus replication. A replication-competent vaccinia virus (GLV-1h68) that also carries marker genes was systemically administered, and resulted in regression of human pancreatic tumor xenografts . An oncoA variant of measles virus with the ability to enter cells efficiently through the CD46 receptor caused a potent cytopathic effect as a result of syncytia formation. It slowed BxPC-3 xenograft tumor growth and extended survival in mice, although it did not completely eradicate the tumors .Pre-clinical studies have demonstrated that treatment with reovirus, an oncolytic virus selectively able to replicate in cancer cells through exploitation of abnormal Ras signaling is non toxic, inhibits the peritoneal dissemination of pancreatic cancer cells after intraperitoneal administration of the virus and is able to decrease the number of liver metastasis from pancreatic cancer after systemic delivery .in vitro studies such as selective tumoral replication of Parvovirus [The continuous growing of the oncolytic virus field as a therapy in pancreatic cancer treatment provides with rvovirus , Sindbisrvovirus  or Mixomrvovirus  that havrvovirus ,102-104.5.Transduction efficiency and selectivity to the target cells constitute major issues towards the development of efficacious and safe pancreatic cancer gene therapy. Many efforts are being conducted to modify current vectors to efficiently and specifically target pancreatic cancer cells.5.1.This strategy is based on the modification of the vector tropism to produce tumor cell-specific infection. Several approaches have been developed to change the vector tropism.Genetic modification of the proteins participating in viral entry: In an adenovirus infection the first step in adenoviral entry involves the binding to CAR receptor. Pancreatic tumor cells express limited levels of CAR and this has encouraged research studies to retarget adenovirus entry through cellular receptors abundant in tumoral cells [in vitro, or upon intratumoral injections. However, the disseminated nature of pancreatic cancer would optimally require the application of the gene targeted vectors through intravascular delivery. In this scenario, Ad-based vectors present some limitations due the inherent hepatic tropism, which excludes CAR entry and uses alternate pathways. Studies on the operating mechanisms in the in vivo liver adenoviral transduction have shown the relevance of adenovirus binding to blood factors and highlighted the role for Ad5 hexon protein interacting with coagulation factor X as a major mediator of in vivo liver transduction [env protein have been tested. One example is Rexin-G, a non-replicative-targeted retroviral vector that incorporates a collagen-binding peptide derived from von Willebrand clotting factor, responsible for guiding platelets to injured tissues where collagen is exposed [al cells ,106. Intal cells ,108. In al cells . Moreoveal cells . All thesduction -114. Opt exposed . Transdu exposed .Pseudotyping or incorporating chimeric fibers. Several studies have shown that adenovirus serotypes 11 and 35 present enhanced infectivity compared to Ad5 in pancreatic cancer [c cancer ,118. Morc cancer ,120. In c cancer .Molecular conjugates that link the vector with specific cellular receptors. This approach has been widely explored to modify adenoviral vectors. It is based on the formulation of bispecific conjugates, bearing one component recognizing a region of the adenoviral vector and the other component specific for a cellular receptor, with the adenoviral vector, generating the retargeted vector. Examples are EGFR-retargeted adenovirus, with a bispecific fusion protein composed of a soluble form of truncated sCAR genetically fused to the EGF ligand (sCAR-EGF). The EGFR-retargeted adenoviruses led to enhanced gene transfer efficiency in pancreatic carcinoma cells [in vitro and in vivo [ma cells . Alterna in vivo ,121. Lip in vivo .5.2.Transcriptional targeting utilizes tumor-specific promoters (TSP) for controlling gene expression in the cancer cell. TSP driving the expression of viral essential genes has been tested for the design of oncolytic adenovirus. Promoters from genes altered in a broad number of tumor types and in pancreatic cancer in particular, such as the cyclooxygenase-2 (COX-2), midkine (MK), E2F1, cancer-specific progression elevated gene-3 promoter (PEG-prom), human telomerase reverse transcriptase (hTERT), urokinase-like plasminogen activator receptor (uPAR) have been used to drive E1 and/or E4 adenoviral genes. All of them have shown to be efficient heterologous promoters controlling viral replication in pancreatic cancer tumors.,122-126.TSPs have also been used in adenoviral vectors to target cytotoxic transgenes into the tumor cells. The TSP carcinoembryonic antigen promoter (CEA), COX-2, MK or the tissue specific insulin promoter have been used to target TK gene expression in pancreatic cancer -129. TheIn the context of the Herpes Simplex viral therapies, the promoter/enhancer sequence of the tumor-associated antigen DF3/MUC1 has been used to regulate the expression of the gamma 34.5 HSV-1 gene to control viral replication preferentially in pancreatic tumors .5.3.in vivo with no effect in normal cells [This is the principle that guided the initial development of oncolytic viruses. Adenovirus, HSV-1 and HSV-2 have been modified in different genes. Deletion of E1B was carried out to generate ONYX-015, an adenovirus unable to replicate in normal cells, but able to replicate in P53 mutated cells . Howeveral cells . Since aal cells . Moreoveal cells .A more sophisticated strategy has been designed around the differential response of normal and tumor cells to viral molecules. Adenoviruses present a mechanism to circumvent the blockade in protein synthesis mediated by PKR phosphorylation of the elongation factor eIF2 that takes place in the cell upon viral infection. Adenoviruses synthesize the RNA molecules  which interact with but do not activate PKR, inhibiting its activation and thus allowing for the synthesis of viral proteins . The ratMutations in the Herpes simplex viruses, HSV-1 or HSV-2 gene sequences have also been generated to confer oncolytic activity. For instance, deletion of the HSV-1 gamma 34.5 gene limited replication in tumor cells, although recent studies have shown some degree of neurotoxicity in normal brain cells . Another6.The location and stage of the tumor will influence the choice of the optimal route of administration to apply. The three major studied routes are: the systemic delivery by intravenous administration specially indicated for pancreatic tumors that present distant metastasis; the intraperitoneal delivery, assessed as a locoregional route to target primary tumors and disseminated peritoneal malignant focus and the intratumoral injection to act against primary tumors .All routes face with difficulties to achieve optimal delivery. In theory, vascular delivery of vectors will lead to a larger distribution of the therapeutic agent within the tumor. Also, it could provide an option for transducing micro-metastatic tumors. However, vascular delivery, too, has been frustratingly inefficient so far. Often blood vessels are confined to the tumor stroma, and therefore several layers of stromal cells must be passed before malignant cells can be reached by vascularly applied therapeutic agents . MoreoveAnother commonly studied method of viral vectors delivery to reach pancreatic tumors is intraperitoneal administration. In this case, the vectors do not interact with the components of the vascular system, which reduces some of the hurdles of systemic delivery. However, the physical barriers to reach the bulk of the tumors are increased ,144.Intratumoral injections also show limitations to reach the bulk of tumoral cells. The high interstitial fluid pressure in the tumors causes a convective flow that can inhibit the passive diffusion of the vector into the tumor ,145. Mor7.A Phase I/II clinical trial of inoperable pancreatic cancer was carried out in a genetically modified-cell based system. Microencapsulated cells carrying the gene CYP2B1 followed by ifosfamide administration were administered to 14 patients, and four regressions and 10 cases of stable disease were reported. Median survival was doubled and one-year survival rate was three-times better than the historic control group ,21.A Phase I/II study has been terminated to evaluate the safety of intravenously administered Rexin-G in locally advanced and metastatic pancreatic cancer refractory to standard chemotherapy. Rexin-G is a retroviral vector bearing a cytocidal dominant negative mutant of human cyclin G1. The authors reported no dose-limiting toxicity, no vector DNA integration and absence of replication-competent retrovirus. Also, no vector-neutralizing antibodies were detected. In terms of tumor response, a dose-response effect between overall survival and Rexin-G dosage was observed, with a 28.6% one year survival at the highest dose tested. Thus Rexin-G was safe, well tolerated and contributed to prolong survival in gemcitabine-resistant pancreatic cancer . ImportaSeveral trials have focused on the use of vaccines made from gene-modified pancreatic cancer cells to help the body build an immune response to kill tumor cells. In some of the studies, vaccines have been administered in combination with chemotherapy. Two trials have been completed, although the results have not yet been published. One of the studies is a Phase I/II study of an antitumor vaccination using alphagalactosyltransferase expressing allogeneic tumor cells engineered by retroviral transduction . Its aim was to establish the proper vaccine dose, examine side effects and potential benefits of the treatment evaluating tumor and immunological responses. The expression of the alphagalactosyltransferase enzyme will result in the incorporation of alpha-gal epitopes on membrane glycoproteins and glycolipids increasing their immunogenicity by triggering a hyperacute rejection response. The other trial is a Phase II study evaluating the safety and efficacy of allogeneic pancreatic tumor cells genetically modified to express the GM-CSF factor with chemoradiotherapy for resected stage I or stage II adenocarcinoma of the pancreas (NCT00084383). Two additional clinical trials based on the same vaccine but with different combined therapies are also ongoing.versus radiation only followed by cell vaccine injection is still ongoing (NCT00868114).Early Phase I/II clinical trials reported the feasibility to apply intratumoral injections of an adenoviral vector carrying the human tumor necrosis factor (TNF)-alpha gene regulated under the control of a radiation-inducible gene promoter TNFerade™  followed by chemoradiation. A recently published case report on a patient demonstrated the capacity of the therapy to shrink the tumor, facilitating later operability to resect the tumor . HoweverA Phase I study based on the use of a recombinant adenoviral vector expressing the TK gene (AdTK) followed by valacyclovir is recruiting patients. There are two arms of treatment. Arm A is for resectable tumors. AdTK will be given before intratumoral surgery by computed tomography (CT) or endoscopic ultrasound (EUS)-guided plus 14 days of valacyclovir pills. A second AdTK dose will be given into the tumor bed at the time of surgery followed by 14 days of valacyclovir. Arm B is for locally advanced tumors. AdTK will be delivered by CT or EUS-guided injection into the tumor previous to chemoradiation and at week three of chemoradiation. Both injections will be followed by 14 days Valacyclovir (NCT00638612).The use of replication-competent viruses is also under clinical trials. The first study was a Phase I/II carried out with repeated intratumoral administrations of ONYX-15 adenovirus by EUS-guided. The trial showed the feasibility and tolerability of the therapy. However its very limited replication capacity compromised its antitumoral effect .A Phase I study combining suicide gene therapy delivered by the replication competent adenovirus expressing the TK and the CD enzymes (Ad5-yCD/mutTKSR39rep-ADP) with chemoradiation therapy is currently recruiting patients with non-metastatic pancreatic adenocarcinoma. A single intratumoral injection EUS-guided followed by three weeks of 5-FC and valacyclovir plus six weeks of capecitabine and 54 Gy radiation will be applied to patients with non metastatic non-resectable tumors. Five dose levels of adenovirus will be tested and the primary endpoint will be toxicity (NCT00415454).A Phase I study is ongoing to assess the safety and effectiveness of intratumoral injections of the oncolytic Herpes simplex virus, OncoVEX GM-CSF into unoperable patients with pancreatic cancer (NCT00402025).Bik gene to patients with advanced pancreatic cancer is being placed (NCT00968604).Regarding non-viral delivery cancer gene therapy, a Phase I dose escalation study to assess the safety and tolerability of a cholesterol liposome-based nanoparticle carrying the pro-apoptotic 8.A broad number of pancreatic cancer gene therapy strategies have been studied and are at different levels of development. For a large group of them promising preclinical data has been generated although a limited number of clinical trials have been carried out, which highlights the difficulties to move from the bench to the clinic. The nature of pancreatic cancer, with a high percentage of cases presenting some level of dissemination at the time of diagnosis, brings the necessity to deliver therapies through loco-regional or systemic routes. In line with this, the development of selective vectors is of great interest, and as presented in the review, is an active area of research, particularly related to oncolytic viruses that are among the most promising candidates to further develop in clinical application. To continue with the generation of more potent and selective antitumoral gene therapies for pancreatic cancer that can have a strong impact in the clinic, the incorporation of the basic knowledge that tumor biology is constantly providing will become fundamental.Recent theories suggest that a small population of cells within some tumors possess the ability to self-renew and proliferate and are thus able to maintain the tumor mass. These cells, which are called tumor-initiating cells or cancer stem cells (CSCs), have been observed to share certain characteristics with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample ,150. CSCStudies to define the signaling networks in PCSCs will be needed to determine which targets are most likely to be effective. One approach to target stem cells is certainly the inhibition of stem cell-associated pathways . Pharmacological studies are beginning to highlight the potential to target such molecules. For the first time, a multimodal therapy, involving the inhibition of two relevant stem cell pathways then SHH and mTOR pathways and additional chemotherapy resulted in a significant depletion of the PCSC pool reducing tumorigenic and metastatic activity, and long-term event-free survival .Because CSCs possess many of the features of normal stem cells, it will be important to determine if such targeting strategies may be effective in tackling CSCs and discriminate from normal stem cells. This would, of course, be a prerequisite for the clinical application of new treatment modalities. We could envision that gene therapy with specific modifications on the vectors, based on CSC characteristics, can further provide a selective control to preserve normal stem cells.Gene therapy may then become a potent weapon contributing to pancreatic cancer treatment, although not necessarily as a unimodal treatment, but in synergy with already existing cancer therapies.


Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency. The observations suggested that ccf DNA might be a potentially valuable prognostic marker for these patients. The similar increase of ccf mtDNA and ccf nDNA in cancers, and in patients after trauma, implies that both the nDNA and mtDNA might be released from the same tissues of origin and by similar mechanisms. Since the total amount of mtDNA per cell is unknown, determining both species in a single reaction would be the most effective and accurate method to compare relative mtDNA quantities with nDNA genome equivalents. Furthermore, the multiplexed assay for simultaneous testing of two parameters can reduce the time consumed by the diagnostic procedures.Eukaryotic cells have nuclear DNA (nDNA) and additional cytoplasmic mitochondrial DNA (mtDNA). It has been demonstrated that cell-free nucleic acids, In our study, we developed a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of mtDNA and nDNA in paired plasma and serum samples. This method is based on a single-step real-time PCR, using a FAM- and a VIC-labelled probe for determining selected mtDNA and nDNA regions of interest. We optimised the multiplex assays for amplifying nDNA and mtDNA simultaneously and efficiently. Since most methods for DNA extraction are established for extracting and purifying nDNA, in our study we also compared the mtDNA and nDNA quantities by using three commercial kits for ccf DNA extraction.Paired plasma and serum samples were obtained from 25 healthy blood donors, with informed consent. The study was approved by the local institutional review board.The 10 mL of peripheral blood samples for coagulant serum and 10 mL of peripheral blood samples for EDTA plasma were taken from blood donors. The blood samples were processed immediately by centrifugation at 1600 g for 10 min. The plasma and serum layers were transferred to new Eppendorf tubes and centrifuged again at maximum speed (16000 g) for 10 min. Plasma and serum samples were divided into aliquots of 400 μL each and stored at -80 °C.Since there are no commercial kits for ccf mtDNA extraction from serum and plasma, we firstly compared three different DNA extraction methods for co-extracting nDNA and mtDNA using plasma and serum samples from five individuals. DNA extraction from the five paired serum and plasma samples was performed using the QIAamp DNA mini kit (QIAGEN) for a first aliquot. For a second aliquot, we used the High pure PCR template preparation kit (Roche Applied Science), and for a third aliquot the automated method with the MagNA Pure LC DNA Isolation Kit – large Volume (Roche Applied Science) and the MagNA Pure LC Instrument. Visually, the automated method with MagNA Pure LC DNA Isolation Kit seemed to yield larger amounts of mtDNA and nDNA, however no significant differences were observed in the quantities using the different commercial kits . Using the automated method, ccf DNA was extracted from each 400 μL plasma and serum sample, and the DNA preparations were eluted in 100 μL elution buffer according to the MagNA Pure LC software.Five μL of DNA elution were used as template for the real-time PCR analysis. For testing nDNA, the GAPDH housekeeping gene was used with forward 5'-CCCCAC ACACATGCACTTACC-3' and reverse 5'-CCTAGTCCC AGGGCTTTGATT-3' primers and 5'-MGB-TAGGAAG GACAGGCAAC – VIC-3' as the probe. For determining mtDNA, a sequence of the MTATP 8 gene starting at locus 8446 was amplified, with forward primer 5'-AATATTAA ACACAAACTACCACCTACC-3', reverse primer 5'-TGGTTCTCAGGGTTTGTTATAA-3' and a 5'-6-FAM-CCTCACCAAAGCCCATA-MGB-3' probe  a known concentration of human genomic DNA with six points ranging from 3.125 x 104 to 10 pg/μL . The latter dilution series showed higher reproducible standard dilution curves than the former, and was therefore used for the further experiments.The efficiency of the multiplex assay for amplifying both nDNA and mtDNA was measured with standard curves generated by dilution series. Two kinds of dilution series were used for the measurements: 1) HPLC-purified single-stranded synthetic DNA oligonucleotides (Microsynth) specifying a 79-bp mtDNA amplicon and a 97 GAPDH amplicon with 6 concentration points ranging from 5 x 10The concentrations of ccf nDNA were estimated according to the standard curves, using the known concentration of human genomic DNA, and were expressed as genome-equivalents (GE) per mL of plasma or serum. A conversion factor of 6.6 pg of DNA per cell was used to calculate the GE :1) 2) nDNA x fold-changemtDNA and expressed also as GE per mL of plasma or serum.Relative quantities of ccf mtDNA could be estimated using an equation of GEData were analysed using the SPSS software . Quantities of ccf mtDNA and ccf nDNA are expressed as median, range and fold change. The Spearman Rank Test was applied to analyse the relationship between mtDNA and nDNA amplifications. Mann-Whitney and Krurkal-Wallis tests were used to determine the statistical significance of the differences between the measured concentrations of nDNA and mtDNA.Ccf DNA extracted by two different manual methods and one automated method was amplified and compared for differences in the quantification of nDNA and mtDNA. Using the three different kits, it was possible to co-extract nDNA and mtDNA from plasma and serum samples. The automated method showed greater advantages, as it proved less time- and labour-consuming, and minimized the risk of contamination, and was therefore used for further experiments.The primers and probes for amplifying GAPDH have been successfully used in our many previous studies, and the specificity of the assay has been confirmed  were obtained using our optimised TaqMan PCR conditions. The two assays on the dilution series were very similar and showed very good correlation to each other and reproducibility. The average correlation coefficient of the 10 standard curves using the Spearman Rank Test was 0.99  CtnDNA-CtmtDNA (R0mtDNA / R0nDNA = (1 + EnDNA)CtnDNA / (1 + EmtDNA)CtmtDNA = (1 + EnDNA)CtnDNA-CtmtDNA, if EnDNA = EmtDNA; (1 + EnDNA)CtnDNA-CtmtDNA 2CtnDNA-CtmtDNA, if E close to 100%).The ccf nDNA equivalents were calculated according to the standardised method, using very reproducible standard dilution curves, which have been described in our previous studies . This may be a result of the release of cellular DNA artefacts during blood clotting procedures. The ccf mtDNA levels in the plasma and serum samples were significantly higher than those of ccf nDNA (9-34 fold).In this study, we described the multiplex assays used to analyse ccf nDNA and ccf mtDNA simultaneously. Using the optimised PCR protocol, the amplification of nDNA and mtDNA in a single reaction tube was very similar and showed a simulation of real-time PCR kinetics. With the comparative efficiencies of two assays in a single tube, we could use the nDNA level as a reference to assess the relative quantities of mtDNA. The method is simple and does not require generating standard curves, which often result in errors due to dilution inaccuracy. Using the multiplex assays, we could rapidly and accurately determine the levels of ccf nDNA and ccf mtDNA in serum and plasma samples. The levels of ccf mtDNA in circulation are significantly higher than those of ccf nDNA, due to the fact that the number of mitochondrial genomes in a cell ranges from several hundreds to more than 10,000 copies, and each mitochondrion contains between two and 10 mtDNA molecules . The serQuantitative alterations of ccf nDNA and mtDNA have been observed in many conditions, especially in aging, apoptosis and carcinogenesis  found up to 612 nuclear integrations. Their homology with the current mtDNA was up to 99%. An accidental co-amplification of these nuclear copies of mitochondrial genes might bias the results (Based on the importance of quantification of both mtDNA and nDNA in life science, we developed a rapid, accurate, simple and low-cost approach that enables the simultaneous identification of physiological and pathogenic mtDNA and nDNA variants. Since there are no commercial kits for ccf mtDNA extraction from serum and plasma samples, we, for the first time, compared three different DNA extraction methods for co-extracting nDNA and mtDNA from plasma and serum. We were also the first ones to examine the efficiencies of the two assays in a single tube. After calculating the comparative efficiencies we could use the nDNA level as a reference to assess the relative quantities of mtDNA, which can simplify the calculation of mtDNA content. The method is simple and does not require generating standard curves, which often result in errors due to dilution inaccuracy. This method can be considered a standard approach for widely quantifying both mtDNA and nDNA in different kinds of samples.


A fundamental question in the emotional memory literature is why emotion enhances memory in some conditions but disrupts memory in other conditions. For example, separate studies have shown that emotional stimuli tend to be better remembered in long-term episodic memory (EM), whereas emotional distracters tend to impair working memory (WM) maintenance. The first goal of this study was to directly compare the neural correlates of EM enhancement (EME) and WM impairing (WMI) effects, and the second goal was to explore individual differences in these mechanisms. During event-related functional magnetic resonance imaging (fMRI), participants maintained faces in WM while being distracted by emotional or neutral pictures presented during the delay period. EM for the distracting pictures was tested after scanning and was used to identify successful encoding activity for the picture distracters. The first goal yielded two findings: (1) emotional pictures that disrupted face WM but enhanced subsequent EM were associated with increased amygdala (AMY) and hippocampal activity  coupled with reduced dorsolateral PFC (dlPFC) activity ; (2) trials in which emotion enhanced EM without disrupting WM were associated with increased ventrolateral PFC activity. The ventral-dorsal switch can explain EME and WMI, while the ventrolateral PFC effect suggests a coping mechanism. The second goal yielded two additional findings: (3) participants who were more susceptible to WMI showed greater amygdala increases and PFC reductions; (4) AMY activity increased and dlPFC activity decreased with measures of attentional impulsivity. Taken together, these results clarify the mechanisms linking the enhancing and impairing effects of emotion on memory, and provide insights into the role of individual differences in the impact of emotional distraction. Emotion is a “double-edged sword” that can either enhance or hinder various aspects of our cognition and behavior. For instance, the emotional charge of an event can lead to better episodic memory (EM) for that event, whereas task-irrelevant emotional distraction can impair working memory (WM), if presented concurrently with goal-relevant information  and a dorsal neural system associated with cold executive processing (ColdEx system)  studies by Dolcos et al. and studies by others  if resource re-allocation by emotional distraction during the WM task coincides with the initiation of processing that also leads to enhanced EM for the distracters themselves, the same AMY regions should play a key role in both of these opposing effects. However, this would produce different effects in brain areas linked to initial WMI (reduced dlPFC activity) vs. long-term EME (increased MTL activity) effects, respectively; (2) coping with emotional distraction would be associated with increased activity in PFC regions. Regarding the second goal, we predicted that individual variations in the susceptibility to emotional distraction would differentially affect the response in emotion and cognitive/executive control brain areas. Specifically, participants with increased susceptibility to WMI would show (3) greater AMY increases and PFC reductions to emotional distraction, and (4) increased AMY activity and decreased PFC activity linked to measures indexing enhanced susceptibility to distraction and impaired executive control.Analyses were performed on data from 17 young (19–35 years of age) healthy right-handed female participants, recruited from Duke University community. We restricted our study to female participants in order to maintain homogeneity of the subject sample, as evidence shows that women and men differ in terms of general emotional reactivity Shields, , responsSubjects performed a combined WM-EM task measuring both the immediate impact of emotion on WM and the long-term impact on EM for the distracters themselves.Subjects were scanned while performing a delayed-response WM task with novel distracters presented during the delay interval between memoranda and probes  or not (New); 50% of the probes were Old and 50% were New. Subjects were instructed to make quick and accurate responses while the probes were on the screen, and then they also rated the level of confidence (LOC) of their responses, using a 3-point Likert scale . The LOC rating was followed by a 10 s inter-trial interval (ITI), to allow the hemodynamic response to return to baseline. The total length of each trial was 26 s.Eight sets of 16 trials  were created and randomly assigned to 8 experimental blocks/runs. To avoid induction of longer-lasting effects, the trials within each run were pseudo-randomized, so that no more than two consecutive trials of the same type were presented. To prevent possible biases resulted from using the same run order, participants were assigned different run orders; a total of 8 different run orders were involved. Each trial started with the presentation of face memoranda (1 s + 1 s), which subjects were instructed to encode and maintain in WM during the delay interval (10 s) between the offset of the second memoranda and the onset of the memory probe. Presentation of the novel picture distracter started 4 s after the offset of the memoranda, and occurred for a total time of 3 s. Participants were instructed to look at the distracters but maintain focus on the WM task, and when the single face probe appeared they had to indicate by a button press whether the face was part of the current memorandum (Old) or not (New). Participants were encouraged to make quick and accurate responses while the picture was on the screen, and then they also rated the LOC of their responses, using a 3-point Likert scale (2 s); the LOC rating was followed by a 2 s ITI.One week following scanning, subjects performed a surprise memory task that tested EM for the emotional and neutral pictures previously presented as distracters during the WM task. The test included 192 pictures  out of which ~2/3 were old pictures. Old and new pictures did not reliably differ in normative intensity scores. All pictures were displayed in black-and-white for increased task difficulty. Trials within each block were pseudo-randomized, so that no more than two consecutive trials of the same type were presented, and participants were assigned different run orders. Each picture was displayed for 3 s and subjects had to indicate by a button press whether the picture was previously seen during the WM task . These ratings were assessed to confirm that the negative distracters were perceived as more emotional than the neutral distracters, and to calculate individual indices of emotional sensitivity to the distracters . Given the possibility that differences in both emotional and cognitive control/executive processing can modulate the relationships between the immediate and long-term impact of emotion, measures indexing general emotion and executive processing were also assessed in participants. Aspects related to general emotional state were assessed using the Positive and Negative Affective Schedule . After localizer, anatomical series, and high-order shimming, functional volumes were acquired using an inverse-spiral pulse sequence . Each volume consisted of 30 functional slices acquired axially (voxels size: 4 mm × 4 mm × 4 mm), thus allowing full-brain coverage. Anatomical scans consisted of high-resolution three-dimensional spin-echo structural images, which were acquired coplanar with the functional slices .Hits = Probes from memoranda (Old) correctly classified as Old, (2) Misses = Probes from memoranda incorrectly classified as New, (3) Correct Rejections (CRs) = New probes correctly classified as New, and (4) False Alarms (FAs) = New probes incorrectly classified as Old. For the EM task, the probes were subsets of the distracters used in the WM task, supplemented with new pictures as foils. Responses in the EM task were classified similarly to the WM task into Hits, Misses, CRs, and FAs. Percentages of probes correctly identified as being Old or New were also calculated for each participant [% Correct = (% Hits + % CR)/2]. Although based on previous studies  were assessed using t statistics and mixed-model ANOVAs. Based on the subjects’ ratings of the emotional intensity of the distracters, individual indices of emotional sensitivity to the present distracters were calculated separately for each subject by subtracting the average of their own ratings for the neutral distracters from the average of their ratings for the emotional distracters. Finally, to assess the role of individual differences in the relationships between behavioral performance and personality, correlations between WM/EM performance and affective/executive measures were also calculated.Responses in the WM task were classified in one of the four categories derived from signal detection theory . For individual analyses, task-related activity was identified by convolving a vector of the onset times of the distracters with a synthetic hemodynamic response and its temporal derivative. The general linear model, as implemented in SPM2, was used to model the effects of interests and other confounding effects . There were 14 first-level regressors: eight task variables  and six motion regressors . Group analyses were conducted using random-effects models to assess the effect of distracter content. The following contrast images were taken to the second level: (1) contrasts linking WM impairment due to emotion and EM for the distracters themselves , (2) contrasts linking WM success in the face of emotional distraction and subsequent EM for the distracters themselves , (3) contrasts linking both WM and EM success, calculated separately for trials with emotional and neutral distracters that were subsequently remembered in the EM task .Imaging data analyses were performed using SPM2 in conjunction with in-house custom MATLAB scripts. Statistical analyses were preceded by the following preprocessing steps: quality assurance, TR alignment, motion correction, coregistration, normalization, and smoothing  identify the neural mechanisms linking the immediate impact of emotional distraction on WM and the EM for the distracters themselves, and to (ii) investigate the role of individual differences in these effects. To accomplish these goals, brain regions in the ventral-affective and dorsal-executive neural systems, specifically sensitive to the presence of emotional distraction were defined as McCarthy  was furtt maps contrasting the emotional and neutral distracters were computed for items that impaired WM and were later remembered: (Emo WM-F and EM-R > Neu WM-F and EM-R), for activity in the ventral-affective network and (Neu WM-F and EM-R > Emo WM-F and EM-R), for activity in the dorsal-executive network. To identify brain mechanism linking the initial effect on WM to the long-term impact on EM in conditions where participants coped with the presence of emotional distraction, we investigated activity for trials in which emotion enhanced EM without disrupting WM. For this, first, t maps contrasting the emotional and neutral distracters were computed for items that were associated with WM success and were later remembered: (Emo WM-R and EM-R > Neu WM-R and EM-R), for activity in regions of the ventral network, and (Neu WM-R and EM-R > Emo WM-R and EM-R), for activity in regions of the dorsal network. Then, to further check whether activity in these regions was also specifically linked to WM success, the maps identifying regions associated with WM success for items that were later remembered, as identified above, were inclusively masked with t maps contrasting activity for items associated with WM success vs. impairment. The latter were separately calculated for trials with emotional and neutral distracters that were subsequently remembered in the EM task: (Emo WM-R and EM-R > Emo WM-F and EM-R), for activity in ventral, and (Neu WM-R and EM-R > Neu WM-F and EM-R), for activity in dorsal regions. The main focus of the present investigation was on identifying the brain regions involved in linking the immediate impact of emotional distraction on WM and the subsequent EM for the distracters themselves. For this reason, the main analysis focused on trials corresponding to distracters meeting both criteria . Activity for these trials was separately identified for the emotional and neutral distracters and then compared to each other. Hence, the link between WM and EM was identified at the level of the trials and the impact of emotion was calculated relative to the neutral stimuli with a similar outcome – i.e., Emo WM-F and EM-R > Neu WM-F and EM-R, for activity in the ventral system, and the reverse contrast for activity in the dorsal system. Importantly, the fact that some participants did not show an overall impairing effect of emotional distraction at the behavioral level did not affect the analyses of the fMRI data, as the trials linking the opposing effect of emotion on WM and EM could be identified in all participants regardless of the overall impact of emotional distraction on WM.Related to our first main goal, to identify brain regions linking the WMI and EME effects of emotion, t-tests. Then, to make sure that regions identified by the between-groups analysis were also sensitive to the effects of emotional distraction, the resulting t maps were inclusively masked with statistical maps identifying a main effect of emotion in the targeted group . For example, increased activity to emotional distraction in the ventral system, in the WMI subgroup was identified by [WMI subgroup (Emo > Neu) > Non-WMI subgroup (Emo > Neu)] ∩ [WMI subgroup (Emo > Neu)]. This more stringent approach ensured that the effect captured by the between-groups comparison came from a difference going in the expected direction in the group of interest, and is not driven by the lack of effects coupled with differences going in opposite direction in the other group. The other main analysis involved identification of brain-behavior relationships by calculating co-variations between the fMRI signal and behavioral and personality measures, to further clarify the significance of effects in brain areas showing differences in activation. The focus was on measures indexing susceptibility to distraction, as reflected in the WM performance and personality scales, such as BIS, which measures a personality trait that has been linked to impaired executive performance. While the stringent masking criteria employed may have offered enough protection against Type I error, given the relatively small sample  with those who did not (Non-WMI subgroup), in response to emotional relative to neutral distraction. This analysis involved a between-samples comparison, to identify differences in brain activity between these two subgroups, in the ventral and dorsal networks. For this, subject-level effects contrasting brain activity for emotional and neutral distracters were first calculated , to be used as input for second level between-groups Yarkoni, , the fina priori defined ventral and dorsal ROIs , activity in the areas identified by the contrasts described above was assessed with a threshold of p < 0.05, uncorrected. This allows direct comparison with a complementary study investigating similar issues in the perceptual domain  = 0.01; p = 0.989]. Although this result was inconsistent with the expected pattern of lower WM performance in response to emotional relative to neutral distracters observed in other previous investigations using similar tasks with emotional distraction  showed the pattern of impaired WM performance to emotional relative to neutral distracters , whereas the remaining ~40% of the subjects (N = 7) did not show it . It should be noted that the fact that some participants did not show an overall impairing effect does not affect the analyses of the fMRI data focusing on the trials associated with WM errors and successful subsequent EM. Regarding the EM performance, as expected, the majority of subjects  remembered better the emotional relative to neutral distracters , and this effect was strongest in trials associated with the highest level of confidence . Further explorations of EM performance revealed a tendency for the participants who showed impaired WM performance to emotional distraction (WMI subgroup) to have better EM for the distracters themselves , although this difference was not statistically significant. A mixed-design ANOVA (WM subgroup × Distracter type) on EM performance confirmed these impressions, yielding a significant main effect of Distracter type , a marginal effect of subgroup , and no interaction effects (p = 0.585). Overall, these results show a differential impact of emotional distraction on WM vs. EM and suggest a link between the initial and long-term effects of distraction.Overall, WM performance was equivalent across both trial types , the WMI subgroup also perceived the negative pictures as more negative relative to the neutral pictures . This was reflected in higher individual indices of emotional sensitivity . In addition, the WMI subgroup also had lower scores in the Self Control subscale of the BIS questionnaire . There were no other differences between the WMI vs. Non-WMI subgroups. Interestingly, correlation analyses showed that the scores for negative general affective state, as assessed by post-WM task PANAS-state, were negatively correlated* with the WM scores for trials with negative distracters . In other words, participants who were affected more by the negative distraction during the WM task also reported more negative emotions following the task.To further elucidate these differential effects of emotional distraction on WM and EM, additional analyses were performed on behavioral and personality data. These analyses revealed that subjects showing systematic impaired WM performance in the presence of emotional distraction (WMI subgroup) also experienced the emotional distracters as more emotional. Specifically, in addition to overall greater ratings for emotional than neutral distracters observed across all subjects [emotional = 6.5; neutral = 2.5; Taken together, the behavioral results identified differential effects of emotion on WM vs. EM, consistent with a link between the initial and long-term effects of distraction, and that these effects were influenced by individual differences. Results from analyses of brain imaging data conducted to investigate the neural correlates of these effects are presented below.WM-F and EM-R > Neu WM-F and EM-R) identified increased activity in the same AMY region linked to both WMI and EME effects  activity  identified increased activity in a right vlPFC region . Thus, individual differences in the susceptibility to emotional distraction were associated with opposing effects in ventral (AMY) and dorsal (dlPFC) regions.Consistent with the behavioral results, fMRI analyses comparing brain activity between participants showing impaired WM performance compared to those who were not impaired by emotional distraction (WMI subgroup vs. Non-WMI subgroup) identified increased AMY activation and dlPFC r = 0.74, p = 0.008; Non-WMI subgroup: r = 0.07, p = 0.44). Thus, individual differences in attentional impulsivity were associated with opposing patterns of co-variation with activity in ventral (AMY) and dorsal (dlPFC) regions.Exploratory analyses were also performed to investigate possible relationships between individual differences in personality measures indexing impaired executive control  and brain activity. These analyses also targeted ventral affective and dorsal-executive regions showing sensitivity to emotional distraction, and where differences in activity were observed between the WMI and Non-WMI subgroups. These analyses revealed opposing relationships between brain activity in response to emotional distraction and individual scores for the AI subscale of BIS, in AMY and dlPFC Figure . SpecifiThe present study used a novel combined WM-EM experimental paradigm to investigate (i) the relationship between the immediate impact of emotional distraction on WM and the long-term impact on EM, and (ii) the role of individual differences in the impact of emotional distraction. The first goal yielded two main findings: (1) emotional pictures that disrupted face WM but enhanced subsequent picture EM were associated with increased AMY and HC activity coupled with reduced dlPFC activity Figure ; (2) trideactivation in dorsal-executive regions   The present results support the idea that the relationship between the immediate impact of emotion on WM and the long-term enhancement of EM is modulated by both direct/bottom-up MTL-based and mediated/top-down PFC-based mechanisms, and that AMY has a central role in both effects. Analyses contrasting the emotional and neutral distracters that impaired WM performance and were later remembered showed that the same AMY region was linked to both of these opposing effects. However, the same trials were associated with opposing modulation of HC (increased) and dlPFC (decreased) activity. These results are consistent with previous investigations linking AMY-HC engagement to a direct route contributing to the memory-enhancing effect of emotion  Turning to our second main findings, increased vlPFC activity for emotional distracters associated with stimuli that did not impair WM but were later remembered provides evidence linking the mechanisms involved in coping with emotional distraction with those involved in enhanced EM. Increased vlPFC activity and AMY-vlPFC coupling have been linked to the engagement of PFC control mechanisms in order to cope with distracting emotions, leading to a diminution of the negative impact of distracters on on-going cognitive processes  The present findings expand previous evidence suggesting individual variation in the response to emotional distraction  Providing further support to the differences in activation, correlation analyses revealed opposing patterns of co-variation of AMY and dlPFC activity with measures of trait AI – i.e., AMY activity increased and dlPFC activity decreased with AI scores. Interestingly, the group-level positive correlation with AI scores in the right AMY was driven by the subjects with increased susceptibility to emotional distraction (WMI subgroup) and overlapped with the left AMY region showing increased response in these subjects when compared to those unaffected by emotional distraction (Non-WMI subgroup). Given the evidence that AI is characterized by increased distractibility and reduced ability to focus attention , associated with bottom-up effects, and higher-level executive regions (dlPFC), associated with top-down influences.hypervigilance along with an overall heightened sensitivity to both threatening and non-threatening stimuli observed in PTSD patients  emotional pictures that disrupted face WM but enhanced subsequent EM were associated with increased AMY and HC activity coupled with reduced dlPFC activity; (2) trials in which emotion enhanced EM without disrupting WM were associated with increased vlPFC activity. Regarding the role of individual differences in the impact of emotional distraction, the study yielded two additional findings: (3) participants who were more susceptible to the WMI effect of emotion showed greater AMY increases and PFC reductions; (4) AMY activity increased and dlPFC activity decreased with measures of attentional impulsivity. Collectively, these findings demonstrate that the immediate impact of emotional distraction on WM and the long-term impact of emotion on EM are mediated by overlapping and dissociable neural systems, involving both ventral/bottom-up and dorsal/top-down mechanisms, and that the brain regions mediating these effects are specifically sensitive to modulations by individual differences. Understanding the mechanisms mediating the impairing and enhancing effects of emotion on cognition, in general, and on memory, in particular, offers potential insights into understanding affective disorders, such as anxiety and depression, where their interaction is The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In July 2012 the U.S. FDA released a statement that manufacturers had voluntarily ceased selling agents containing the compound C8 for food-contact use the prior summer.Testing the Waters report on water quality at U.S. ocean and Great Lakes beaches.In June 2012 the Natural Resources Defense Council released its annual The Future We Want,In adopting the consensus document A new study employing satellite-based wildfire data from the past decade and 16 different global climate change models (GCMs) estimates that the likelihood of fires like the summer 2011 events in western United States will particularly increase in mid- and high-latitude areas of the Earth in the next 30 years.It has been difficult to image particulate matter (PM) without introducing artifacts caused by capturing particles for microscopic analysis. Now researchers have found a way to do so using the Linac Coherent Light Source free-electron laser, the world’s most powerful X-ray laser.


The manner in which different distributions of synaptic weights onto cortical neurons shape their spiking activity remains open. To characterize a homogeneous neuronal population, we use the master equation for generalized leaky integrate-and-fire neurons with shot-noise synapses. We develop fast semi-analytic numerical methods to solve this equation for either current or conductance synapses, with and without synaptic depression. We show that its solutions match simulations of equivalent neuronal networks better than those of the Fokker-Planck equation and we compute bounds on the network response to non-instantaneous synapses. We apply these methods to study different synaptic weight distributions in feed-forward networks. We characterize the synaptic amplitude distributions using a set of measures, called tail weight numbers, designed to quantify the preponderance of very strong synapses. Even if synaptic amplitude distributions are equated for both the total current and average synaptic weight, distributions with sparse but strong synapses produce higher responses for small inputs, leading to a larger operating range. Furthermore, despite their small number, such synapses enable the network to respond faster and with more stability in the face of external fluctuations. Neurons communicate via action potentials. Typically, depolarizations caused by presynaptic firing are small, such that many synaptic inputs are necessary to exceed the firing threshold. This is the assumption made by standard mathematical approaches such as the Fokker-Planck formalism. However, in some cases the synaptic weight can be large. On occasion, a single input is capable of exceeding threshold. Although this phenomenon can be studied with computational simulations, these can be impractical for large scale brain simulations or suffer from the problem of insufficient knowledge of the relevant parameters. Improving upon the standard Fokker-Planck approach, we develop a hybrid approach combining semi-analytical with computational methods into an efficient technique for analyzing the effect that rare and large synaptic weights can have on neural network activity. Our method has both neurobiological as well as methodological implications. Sparse but powerful synapses provide networks with response celerity, enhanced bandwidth and stability, even when the networks are matched for average input. We introduce a measure characterizing this response. Furthermore, our method can characterize the sub-threshold membrane potential distribution and spiking statistics of very large networks of distinct but homogeneous populations of 10s to 100s of distinct neuronal cell types throughout the brain. Circuits of the MindExperiments analyzing the distribution of synaptic weights impinging onto neurons typically observe low-amplitude peaks with only few, large-amplitude excitatory (EPSPs) or inhibitory post-synaptic potentials (IPSPs) The relation between probabilistic synaptic weight distribution and population dynamics can be studied using simulations The equation for the membrane potential probability current injection of the neuron which previously spiked. The first term on the RHS in The stationary solution for Although the model can include both excitatory and inhibitory connections, all the results presented below, except for those in Intuitively, this model quickly diverges from the Fokker-Planck formalism when the synaptic strength is large. Consider the hypothetical case of a neuron starting at rest which has all the synapses equal and large. After a short time step, the Fokker-Planck formalism produces a narrow Gaussian distribution in membrane potential near rest, while the DiPDE formalism yields a membrane potential distribution which is a sum of two scaled delta functions: a large one at rest, and a small one at the synaptic weight. Over time, the Fokker-Planck equation converges to a single broader Gaussian, while the DiPDE formalism leads to a larger coefficient for the delta function at the synaptic weight value. A generalization to conductance-based synapses is presented in  synaptic weight and input rate , so that the average synaptic current was the same see . We alsoon input , reflectSince the network we study is a feed-forward network, the mean synaptic delay results in a simple time translation of the responses. However, the overshoot of steady state seen in tail weight numbersTo analyze what characteristic of the synaptic distribution is most important for the fast response observed for our heavy-tailed distributions, we generated more than 1000 random distributions matched to have the same mean synaptic weight and input rate . Higher Having established a measure of population activity when all neurons start at rest, we examined the dynamics resulting from an equilibrium different from rest , which is known to play a key role in neural network homeostasis and in the generation of multiple network states  Hz . Heavier Hz .In the preceding analysis, we saw how perturbations of the input event rate in a population with different synaptic weight distributions affect the entire time-course of the population's evolution from an initial to a final equlibrium. In contrast, we also analyzed how fluctuations in inputs see .additional inputs on average. This means that, effectively in a time To quantify the instantaneous change in the equilibrium firing rate, we make use of a closely-related variant of the tail weight numbers defined in Using A few strong synapses can exert an undue large influence on the mean response of the population. Consider the unimodal  is  see .Our study provides an advance on two fronts, computationally and neurobiologically. First, we developed and validated a semi-analytic method to model the sub-threshold membrane potential probability distribution and the firing rate of homogeneous neuronal populations with finite synaptic inputs. Second, we apply this method to explore the effect of varying synaptic weight distributions on equilibrium and transient population characteristics. From a methodological standpoint, the DiPDE formalism reproduces population behavior from aggregate simulations of identical point neurons, without the need to run the large-scale simulations themselves with the attendant computational costs see . The Fokpopulation statistics method makes possible the modeling and characterization of the membrane potential distribution and the spiking statistics of very large networks of distinct but homogeneous populations of hundreds of neuronal cell types in numerous brain regions. This would be relevant, for instance, when modeling the resting state activity at the cellular level throughout the awake or the sleeping brain. Second, in order to understand the biological significance of powerful but rare EPSPs This Teramae and colleagues in vivo onto the critical role that such rare, “Black Swan”-like, events can play in the day-to-day life of the brain.Such investigations highlight the need to focus the attention of electrophysiologists studying synaptic transmission in vivo conditions, distinguishing between these distributions in practice would not be easy as it would require recordings of long duration to collect the relevant statistics. For experimental purposes, the numbers of independent recordings of sub-threshold steady state membrane potential required to differentiate between different synaptic weight distributions have been provided in Given the large biological and instrumental noise present in synaptic measurements, in particular under A simplified case can be obtained if all EPSPs are assumed to have the same value If In If Numerical simulations, against which the Fokker-Planck (for current-based synapses) and DiPDE formalisms were compared, were performed by invoking the NEST simulator after writing the code in PyNN.For current-based synapses, simulations were performed for a population of For conductance-based synapses, simulations were performed for a population of Simulations were also performed for a population of To solve the evolution For the standard LIF neuron with membrane time-constant For more general forms of the leak term  neuron; see At each time-step In order to compare results with simulations, we need to match the total synaptic input to the neuronal population with that from simulations. The DiPDE formulation is exact when implementing instantaneous synapses The synaptic weight For our choice of simulation parameters outlined above The underlying stochastic process in Expected 95% intervals for output spike counts can then be obtained as follows. At each time step, the solution obtained from For non-instantaneous synapses, upper and lower bounds on the output firing rate can be obtained. The exponentially decaying current-based synapses used here in simulations ) result in an exact EPSP given by Setting A lower bound on the output firing rate can obtained by equating the maximum depolarization, For For the lower bound, consider the difference between For current-based synapses, the change in membrane potential resulting from synaptic input is independent of the initial membrane potential. For a conductance-based synapse however, this change is proportional to the difference between the initial membrane potential and the reversal potential Because of the additional dependence on the membrane voltage and synaptic reversal potential For our choice of simulation parameters, the maximum depolarization achieved by a neuron starting from rest due to synaptic input with For multiple neuronal populations connected to each other, one can generalize The DiPDE formalism  can be gFor the exponential integrate-and-fire (EIF) neuron, the numerical solution of the equation with exponentially decaying conductance-based synapses gives The Fokker-Planck equation for current-based synapses was solved using an explicit Forward-Time Centered-Space (FTCS) scheme. As in the DiPDE, the leak term was solved analytically, although the voltage discretization was kept uniform in order to be compatible with the standard Centered-Space discretization used for the diffusion term. To ensure that the discretization was sufficiently fine , we compared the equilibrium firing rate generated by this scheme against the analytical solution The effect of various matched synaptic weight distributions on the population dynamics in feed-forward networks was investigated using the DiPDE formalism. The top panel of If the firing is driven exclusively by variations in input , the differences in output firing rates are large as shown in the bottom panel of These results imply that population response is determined not only by the total current, but also by the mean synaptic weights.For the bottom row of Delta : Gaussian : Exponential : Lognormal : Bimodal : Power-law : 2), Gaussian (1.3 mV2), Exponential (1.8 mV2), Lognormal (2.2 mV2), Bimodal (8.0 mV2) and Power-law (8.7 mV2). The firing rates and transient times corresponding to distributions for which the mean input current was matched , the output synaptic weight distribution from that population to the target population is convolved with the synaptic utility distribution,To analyze the effect of fluctuations generated by different synaptic distributions, the DiPDE numerical solution was evolved with a given synaptic distribution for 300 ms, long enough for the voltage distribution to reach equilibrium. At this point, to simulate fluctuations which occur on timescales This same analysis was then conducted with synaptic depression during the fluctuation stage, starting with the stationary distribution obtained without synaptic depression. Unlike in the no-depression case, the synaptic distribution was scaled by the synaptic utility  simulations of 10,000 leaky integrate-and-fire neurons, b) the numerical solution to (EPS)Click here for additional data file.Figure S2Solutions to (EPS)Click here for additional data file.Figure S3Comparisons for excitatory conductance-based synapses. Top panels show time evolution of the probability distribution of membrane potentials in the neuronal population obtained from a) simulations, and b) numerical solution to (EPS)Click here for additional data file.Figure S4For non-instantaneous synapses, estimates of the output firing rate can be obtained by using instantaneous synapses equated for the total charge  or the maximum depolarization respectively. These are controlled by an additional parameter (EPS)Click here for additional data file.Figure S5EPSPs from instantaneous current-based, excitatory synapses used for obtaining estimates of the output firing rate for non-instantaneous synapses (see (EPS)Click here for additional data file.Figure S6Gaussian distributions of instantaneous synaptic weights with the same mean input current. Top Panels: a) Four Gaussian synaptic weight distributions. b) The output firing rates as a function of time when the four Gaussian synaptic inputs are activated with an input firing rate adjusted such that the mean input currents are equal. Both low-amplitude distributions (red and light-blue curves) have twice the input rate of the high-amplitude ones (dark-blue and green curves). In the absence of a threshold, the synaptic input would depolarize (EPS)Click here for additional data file.Figure S72/ms. b) Steady state sub-threshold membrane potential distributions. c) Output firing rates. Heavier-tailed distributions still produce quicker transients, but result in lower steady state output firing rates in contrast to  Semi-log plot of different synaptic distributions, matched for drifttrast to .(EPS)Click here for additional data file.Figure S8Effect of (EPS)Click here for additional data file.Figure S9Top Panels: Tail weight numbers (EPS)Click here for additional data file.Figure S10Time taken to reach 20% of steady-state firing rate ((EPS)Click here for additional data file.Figure S11Even (EPS)Click here for additional data file.Figure S12Quantifying response to sudden changes in input rate for different synaptic weight distributions matched for mean input current. Peak output firing rates for different fractions of base input rate as a function of base input rate for a) (EPS)Click here for additional data file.Figure S13Effect of external fluctuations on sub-threshold membrane potential distribution and excitability for different synaptic weight distributions, without and with synaptic depression. Simulations start from the equilibrium of (EPS)Click here for additional data file.Figure S14Effects of fluctuations in synaptic input, starting from the equilibrium obtained for different synaptic weight distributions with mean = 1 mV and input rate (EPS)Click here for additional data file.Table S1Computational times. Table shows the simulation times in (s) with NEST and DiPDE for different choices of time step dt. The middle four columns correspond to simulation times with different numbers of neurons (PDF)Click here for additional data file.Table S2Differentiating synaptic weight distributions matched for mean input current. Table shows the number of independent recordings of sub-threshold steady state membrane potential required to differentiate between synaptic distributions matched for mean input current. The values above the diagonal are the sample sizes needed for p = 0.01, and below the diagonal are the sizes for p = 0.05.(PDF)Click here for additional data file.Table S3Differentiating synaptic weight distributions matched for drift and diffusion. Table shows the number of independent recordings of sub-threshold steady state membrane potential required to differentiate between synaptic distributions matched for drift and diffusion. The values above the diagonal are the sample sizes needed for p = 0.01, and below the diagonal are the sizes for p = 0.05.(PDF)Click here for additional data file.Table S4Dynamical ranges. Table shows the upper (90%) and lower (10%) limits of steady state output firing rates and dynamical ranges (ratio of upper and lower steady state output firing rates) for synaptic weight distributions as in  matched (PDF)Click here for additional data file.Table S5Relative excitabilities. Table shows the maximum relative excitability for synaptic weight distributions matched for mean input current, without and with synaptic depression. Heavier-tailed distributions lead to smaller changes in relative excitability.(PDF)Click here for additional data file.Text S1A brief review of stochastic processes focusing on elements needed for this study.(PDF)Click here for additional data file.Text S2Comparison of DiPDE and Fokker-Planck formalisms.(PDF)Click here for additional data file.Text S3Computational complexity with DiPDE.(PDF)Click here for additional data file.Text S4p-values for differences between sub-threshold steady-state membrane potential distributions obtained from different synaptic weight distributions.(PDF)Click here for additional data file.


About 2% of all paragangliomas are located in the chest, and a few have been described to be found in the heart. Primary cardiac paragangliomas are extremely uncommon tumors and surgical experience with this neoplasm is limited. Treatment strategies described in the literature have included simple excision, excision with reconstruction, autotransplantation after excision of the tumor and even orthotopic cardiac transplantation, depending on the extent of disease. A primary retrocardiac paraganglioma catecholamine-productive was identified in an asymptomatic 49–year old female associated to familial pheochromocytoma-paraganglioma syndrome caused by germline mutation of the gen which codifies for the subunit B of succinate dehydrogenase enzyme (SDHB). The neoplasm was surgically excised from the posterior surface of the left atrium via median sternotomy using cardiopulmonary bypass. Direct ligation of feeding vessels of the tumor along with left atrial reinforcement using a pericardial patch was performed. The post-operative course was uneventful, with normalization of catecholamine secretion and no recurrence at three-month follow-up. We review the current literature about this exceptional cardiac tumor, pathophysiological conditions and options for surgical management. Pheochromocytomas are catecholamine-producing neuroendocrine tumors arise from the chromaffin cells of the embryonic neural crest. It is estimated that the annual incidence is approximately 0.8 per 100.000 persons and they occur most often in the fourth to fifth decade [About 85–90% of them develop in the abdomen  and only 10% originates from extra-adrenal sites. Extra-adrenal pheochromocytomas  originate in the ganglia of the sympathetic nervous system and most are located within the abdomen , head and neck , urinary bladder (1%) and mediastinal compartments (less than 1%) .Thoracic paragangliomas are the most uncommon location and they can be divided into two groups: those located in the anterior mediastinum (arise from parasympathetic paraganglia) and located in the posterior mediastinum (sympathetic chain). Primary cardiac pheochromocytomas (paragangliomas) are extremely rare, occurring in only 0.001%–0.003%, with female predominance [In general terms, the clinical manifestations of these tumors are varied, not particularly specific and it depends on the secretion of excessive amounts of catecholamines, such as noradrenaline (norepinephrine) and adrenaline (epinephrine). Although these tumors may be a cause of hypertension, they are the underlying cause of only about 0.01% of cases of high blood pressure.About 75% of pheochromocytomas are sporadic and the remaining 25% are hereditary. Mutations of the genes which codify for the subunits D, A, C y B of succinate dehydrogenase enzyme  involved in the Krebs cycle can lead to tumorogenesis in chromaffin cells and they have been identified as causing familial adrenal pheochromocytoma and extra-adrenal paraganglioma; moreover, these tumors may also occur in the multiple endocrine neoplasia (MEN) syndrome type 2A and 2B, von Hippel-Lindau disease or von Recklinghausen’s neurofibromatosis .The diagnosis of these tumors can be established by measuring catecholamines and metanephrines in plasma or 24-hour urine along with imaging techniques such as computed tomography (CT), magnetic resonance imaging, iodine-123-marked metaiodobenzylguanidine (MIBG) scan and positron emission tomography (PET) scan .Treatment of pheochromocytomas involves blocking the effect of catecholamines with an alpha adrenergic blocker and after, it then becomes safe to surgically remove the tumor. Although surgical resection is the treatment of first choice in most part of cases, extra-adrenal localizations (such as those paragangliomas originated in cardiac structures) involve an uncommon and complex surgical approach for a complete removal.We describe a case of surgical excision of a primary functioning retrocardiac paraganglioma associated with hereditary syndrome and we review the relevant literature about the peculiar features and surgical strategies for removal of this cardiac neoplasm.We report a case of an asymptomatic 49-year old Caucasian female, with hereditary type 4 pheochromocytoma-paraganglioma syndrome caused by germline mutation  of the gen which codifies for the subunit B of succinate dehydrogenase enzyme (SDHB) of the mitochondrial complex II. She had a family history of extra-adrenal paraganglioma: genetic testing had revealed the gen SDHB mutation for eight first- and second-degree relatives and carotid body paraganglioma had been resected in three of them. Urinary and plasma levels of catecholamines  and their metabolites (normetanephrine) were elevated and computed tomography of the abdomen ruled out adrenal gland involvement.18F-dihydroxy-phenyl-alanine (18F-DOPA) PET scan showed a primary retrocardiac mass with metabolic activity. Metastasic disease was not found and no other intrathoracic masses were identified. Transesophageal echocardiography (TOE) and TC imaging of the heart showed the large mass (40 × 20 mm), extrinsic to the heart, on the posterior wall of the left atrium (LA)  was instituted before operation for the catecholamine-secreting tumor. She underwent surgical removal of the retrocardiac tumor via median sternotomy. As part of our initial surgical planning, cardiopulmonary bypass (CPB) was started through right femoral artery and venous cannulation  Figure A-B becauDirect ligation of feeding vessels was performed for control of bleeding: the collaterals vessels from the right coronary artery (RCA) and circumflex artery (CxA) were ligated Figure C-D. TOE Following excision of the superior pericardial reflection Figure A, the leCPB time was 143 minutes and aortic cross-clamping time was 105 minutes. Intraoperative TOE revealed a complete excision of the tumor and no perioperative hypertensive crisis on palpation of the tumor were demonstrated. The patient was then weaned from bypass successfully.Histological examination confirmed the paraganglioma and malignant changes were not found Figure B-C. Chroth post-operative day and short-term anticoagulation was started for prevention of LA thrombus formation due to transient endothelial dysfunction. At 3–month follow-up, she remains asymptomatic and biochemical evaluations have confirmed the normalization of levels of catecholamines.The post-operative course was uneventful, no bleeding was detected, sinus rhythm was maintained and antihypertensive therapy was not necessary. She was discharged at 8Cardiac paraganglionic tumors are rare neoplasms derived from the neural crest in the mediastinum, associated with the autonomic nervous system, which account for 0.3% of all mediastinal neoplasms and less than 1% of all primary cardiac tumors . They arEdwin Besterman  was the first author to report a successful resection on CPB of a cardiac pheochromocytoma in the LA in 1974 ,9 and thClinical presentation depends on the functional status of the paraganglioma and the location at the heart: paroxysmal hypertension due to secretion of catecholamines, chest pain , symptomIn a short number of patients, cardiac paragangliomas can be associated to hereditary syndromes such as the Carney triad   or familMost cardiac paragangliomas are benign, but local invasion can occur and metastases have been reported in isolated cases ,21. LocaThe preoperative goals for this type of tumors are to normalize hemodynamic variables such as blood pressure and avoid surges in catecholamine release with tumor manipulation during surgery. Nevertheless, preoperative alpha-1-adrenoceptor antagonist seems to have no benefit in maintaining intraoperative hemodynamic stability in patients with normotensive adrenal pheochromocytoma . AlthougLess than 40 cases about surgical excision of a cardiac paraganglioma have been described in adults  and the Because they are highly vascular, some groups advocate the embolization before surgery to reduce perioperative bleeding  when surNevertheless, in our patient, for prevention of a catastrophic haemorrhage during the time of the operation, a direct ligation of feeding vessels of the tumor was performed. To date, isolated cases of direct ligation at surgery have been described  and no cAs these tumors are of locally invasive nature and they have a less well-defined capsule than adrenal pheochromocytomas, a complete resection can be technically difficult by the position of the tumor and should be performed on CPB and cardioplegic arrest in the most part of cases in order to achieve disease-free excision margins. When the interatrial septum is involved, the anterior aspect of the septum must be preserved to avoid heart block; in other situations, the tumor invades surrounding vascular structures and resection may be extremely difficult. For selected patients, extensive cardiac reconstructive surgery may therefore be required, with its associated risks. Our patient underwent a successful complete resection of the “caged” tumor between the pulmonary veins along with reconstruction of the resected site and no complications were detected after surgery.Reconstruction of the LA using either autologous or bovine pericardial patch has been previously described and, when a portion of the pulmonary veins is also infiltrated and excised, it must be repaired and reconstructed with the patch , althougAlthough surgical resection and reconstruction is the gold standard treatment for these tumors, this approach can be impossible due to extensive involvement of the great vessels or other structures of the heart. When total surgical resection is extremely difficult, cardiac autotransplantation has been described and, in selected cases, when a complete removal is not possible, orthotopic cardiac transplantation is a feasible technique in those paragangliomas that have direct coronary artery involvement, extend into the left ventricle or invade the atrioventricular groove . In geneIn the only reported case series including 14 patients over the last 30 years, at Mayo Clinic [When a complete removal of the tumor is achieved, long-term prognosis seems to be excellent and survival of up to 14 years has been reported in a patient with excised mediastinal paraganglioma . To dateOur case is relevant in that there were no classic signs of catecholamine excess despite to be a functioning tumor and it illustrates successful management of a benign left atrial primary paraganglioma associated with SDHB mutation, including direct ligation of feeding vessels of the tumor, complete excision and subsequent reinforcement of the LA, without evidence of recurrence at short-term follow-up.– Primary cardiac paragangliomas are exceptional tumors and surgical resection is the gold standard treatment.– Control of feeding vessels, complete resection of the tumor and reconstruction of the resected sites are the goals for surgical management.– Although treatment strategies can be varied according to the extent of the disease, in general terms, cardiac paragangliomas can be safely resected using well-established surgical techniques with appropriate surgical planning.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.18F-dihydroxy-phenyl-alanine positron emission tomography scan;TOE: Transesophageal echocardiography;LA: Left atrium;CPB: Cardiopulmonary bypass;RCA: Right coronary artery;CxA: Circumflex artery;SVC: Superior vein cavae;RA: Right atrium;Ao: Aorta;CS: Coronary sinus;LPVs: Left pulmonary veins;IVC: Inferior vein cava;LIPV: Left inferior vein cava;LAA: Left atrial appendage;OS: Oblique sinus;H & E: Haematoxylin and eosin;C & S: Chromogranin and synaptophysinSDHB: Subunit B of succinate dehydrogenase enzyme;SDHD: Subunit D of succinate dehydrogenase enzyme;SDHA: Subunit A of succinate dehydrogenase enzyme;SDHC: Subunit C of succinate dehydrogenase enzyme;MEN: Multiple endocrine neoplasia syndrome;CT: Computed tomography;MIBG – scan: Iodine-123-marked metaiodobenzylguanidine – scan;PET – scan: Positron emission tomography – scan;(18F-DOPA) PET scan: The authors declare that they have no competing interests.MTGL: Design, data collection, photographs and writing article. SGG; ESG; SGR; and JGL: Critical revision and helped to draft the manuscript. All authors read and approved the final manuscript.


Rimicaris exoculata dominates the faunal biomass at many deep-sea hydrothermal vent sites at the Mid-Atlantic Ridge. In its enlarged gill chamber it harbors a specialized epibiotic bacterial community for which a nutritional role has been proposed.The shrimp Desulfocapsa. The association of these phylogenetic groups with the shrimp was confirmed by fluorescence in situ hybridization. Based on functional gene analyses, we hypothesize that the Gamma- and Epsilonproteobacteria are capable of autotrophic growth by oxidizing reduced sulfur compounds, and that the Deltaproteobacteria are also involved in sulfur metabolism. In addition, the detection of proteobacterial hydrogenases indicates the potential for hydrogen oxidation in these communities. Interestingly, the frequency of these phylotypes in 16S rRNA gene clone libraries from the mouthparts differ from that of the inner lining of the gill chamber, indicating potential functional compartmentalization.We analyzed specimens from the Snake Pit hydrothermal vent field on the Mid-Atlantic Ridge by complementing a 16S rRNA gene survey with the analysis of genes involved in carbon, sulfur and hydrogen metabolism. In addition to Epsilon- and Gammaproteobacteria, the epibiotic community unexpectedly also consists of Deltaproteobacteria of a single phylotype, closely related to the genus Rimicaris exoculata from the Snake Pit hydrothermal vent field, and suggest that autotrophic carbon fixation is contributing to the productivity of the epibiotic community with the reductive tricarboxylic acid cycle as one important carbon fixation pathway. This has not been considered in previous studies of carbon fixation and stable carbon isotope composition of the shrimp and its epibionts. Furthermore, the co-occurrence of sulfur-oxidizing and sulfur-reducing epibionts raises the possibility that both may be involved in the syntrophic exchange of sulfur compounds, which could increase the overall efficiency of this epibiotic community.Our data show the specific association of autotrophic bacteria with  Rimicaris exoculata2R. exoculata harbors specialized epibiotic bacterial communities on the inner lining of the wall of its expanded branchial chamber, the so-called branchiostegites, and on its modified mouthparts, the scaphognathites and expodites of the first maxillipeds Highly specific epibiotic associations between bacteria and invertebrates are a very common phenomenon at deep-sea hydrothermal vents, contributing to overall biomass production at these sites R. exoculata shrimp at vent sites where the hydrothermal fluids contain high iron, methane, and hydrogen concentrations, and relatively low sulfide concentrations, like for example the Rainbow vent site It has been proposed that the shrimp aggregate in the mixing zone of hydrothermal fluids and ambient deep-sea water to supply the epibionts with both oxidants and reductants to drive chemoautotrophy R. exoculata epibiotic community from the Snake Pit vent site was composed exclusively of one phylotype belonging to the Epsilonproteobacteria R. exoculata individuals from the Snake Pit hydrothermal field by combining a 16S rRNA gene based diversity assessment with a survey of metabolic genes involved in carbon, sulfur, and hydrogen metabolism. In addition, fluorescence in situ hybridization was performed to verify the presence of the detected phylotypes and their location.In a pioneering study, Polz and Cavanaugh R. exoculata, 16S rRNA gene clone libraries were constructed from four different individuals. Two clone libraries originated from DNA extracted from the branchiostegites of individuals 1 (113 clones) and 2 (82 clones), respectively. A third clone library was constructed from the DNA extracted from both the branchiostegites and mouthparts of individual 3 (25 clones), while the fourth and fifth libraries were constructed with DNA extracted from the mouthparts (67 clones) and branchiostegites (64 clones) of individual 4, respectively , representing 49% of the total recovered sequences, belonging to the Marine Group 1 (MG1) cluster within the  cluster . The domDesulfocapsa sulfoexigens and Desulfocapsa thiozymogenes, bacteria capable of disproportionating partially oxidized sulfur compounds Deltaproteobacteria constituted a large fraction of the observed clones (32%), in particular on the branchiostegites . All 111R. exoculata from four vent sites on the MAR Leucothrix mucor, a chemolithoheterotrophic sulfur oxidizer Bacteroidetes were most similar to sequences previously recovered from R. exoculata from three vent sites on the MAR Gammaproteobacteria constituted a small, but consistent fraction of the recovered clones . Only onR. exoculata individual from Snake Pit generally reflected the results of the 16S rRNA gene clone libraries from the mouthparts  of sections through the mouthparts of an uthparts . A specicbbL) and II (cbbM), as well as primers that target fragments of the small (aclB) and large (aclA) subunits of ACL.To assess the autotrophic potential and the biosynthetic pathways involved in carbon fixation of the epibiotic community we constructed clone libraries for genes encoding for subunits of key enzymes of two carbon fixation pathways, RubisCO for the Calvin-Benson-Bassham (CBB) cycle and the ATP-dependent citrate lyase (ACL) for the reductive tricarboxylic acid (rTCA) cycle. The CBB cycle is known to be used for carbon fixation by a number of autotrophs including gammaproteobacterial chemoautotrophs aclA and aclB coding for the two subunits of ATP citrate lyase were detected in the clone libraries from both the mouthparts and the branchiostegites. All acl sequences belonged to a clade containing only sequences from Epsilonproteobacteria , a key gene for the oxidation of reduced sulfur compounds and the reduction of sulfate, could be amplified. AprA sequences related to sequences from gammaproteobacterial sulfur oxidizers were mainly amplified from the mouthparts, whereas sequences related to those of deltaproteobacterial sulfate-reducers, were almost exclusively amplified from the branchiostegites  sequence types related to gammaproteobacterial aprA sequences. These clustered together with clone SCAPS85 of an R. exoculata specimen from Rainbow and clone C5 from the Yeti crab Kiwa hirsutaDesulfocapsa sulfoexigens and Desulfocapsa thiozymogenes , which was further confirmed by FISH. In the present study, we were able to confirm the dominance of OTU S1-4 on the scaphognathites by using a FISH probe specifically targeting this phylotype, but not the other epsilonproteobacterial sequences found in our clone libraries  were retrieved during cruise AMK-47 of the R/V 'Akademik Mstislav Keldysh' in 2002. The geology and geochemistry of the site has been described previously 5′-CTTTCTGGGATTRGCTTGCTCT-3′) 5′-CTGTCGGATTCTCTCAAT-3′) FISH of whole scaphognathite tissues from an individual not used for the preparation of the clone libraries was exactly carried out as previously described aclA and aclB) were amplified using the primer sets F2/R5 (aclA) and 892F/1204R (aclB) cbbLF/cbbLR and cbbMF/cbbMR were used soxB and aprA gene were amplified using the primer sets soxB432F/soxB1446B and aps1F/aps4R, respectively hynL gene were amplified with the primer set hynS330F/hynL419R Genomic DNA was extracted from four shrimp individuals. The shrimp were dissected under sterile conditions and DNA from the epibiotic community on the branchiostegites and/or on the modified mouthparts was isolated using the “PowerSoil DNA extraction kit”  according to the provided protocol. Bacterial 16S rRNA gene fragments were PCR-amplified in eight parallels in a 20-cycle PCR at an annealing temperature of 50°C with the general bacterial primer set 8F and 1492R The pooled amplified PCR products were gel-purified using the QIAGEN QIAquick gel extraction kit  and cloned into pCR4-TOPO plasmid vectors with the TOPO-TA cloning kit  as described by the manufacturer. An environmental clone library for each gene was constructed . Coloniewww.arb-home.de) using the ARB FastAligner utility All sequences were edited with ChromasPro c.c1.33 and compared to the NCBI database using BLAST The sequence data have been submitted to EMBL/GenBank/DDBJ databases under accession numbers FN658693-FN658702, FN659777-FN659803 and FN661670.Table S1Functional gene clone library results.(PDF)Click here for additional data file.Figure S1aclB sequences. The tree was calculated using the Neighbor-Joining method. Bootstrap values are shown as percentages of 1000 bootstrap replicates. Sequences obtained in this study are depicted in red. Scale bar represents 5% estimated sequence divergence.Phylogenetic tree based on translated (TIF)Click here for additional data file.Figure S2cbbM sequences. The tree was calculated using the Neighbor-Joining method. Bootstrap values are shown as percentages of 1000 bootstrap replicates. Sequences obtained in this study are shown in yellow. Scale bar represents 10% estimated sequence divergence.Phylogenetic tree based on translated (TIF)Click here for additional data file.Figure S3hynL sequences. The tree was calculated using the Maximum-Likelihood method. Bootstrap values are shown as percentages of 100 bootstrap replicates. Sequences obtained in this study are highlighted with colors. Scale bar represents 20% estimated sequence divergence.Phylogenetic tree based on translated (TIF)Click here for additional data file.