In situ detection of reactive oxygen species (ROS) using dihydroethidium fluorescence revealed that increased ROS detected in OX-42 positive cells following ischemia was reduced in apocynin-treated rats by ~ 51%, but surprisingly increased in surrounding NeuN positive cells of the same rats by ~ 27%, in comparison to the contralateral hemisphere. Reduced ROS from activated microglia/macrophages treated with apocynin was associated with reduced Nox2 immunoreactivity without change to the number of cells. These findings confirm the protective effects of apocynin and indicate a novel mechanism via reduced Nox2 expression. We also reveal compensatory changes in neuronal ROS generation as a result of Nox2 inhibition and highlight the need to assess long term individual cell responses to inhibitors of oxidative stress.NADPH oxidase is a major source of superoxide anion following stroke and reperfusion. This study evaluated the effects of apocynin, a known antioxidant and inhibitor of Nox2 NADPH, on neuronal injury and cell-specific responses to stroke induced in the conscious rat. Apocynin treatment (50 mg/kg i.p.) commencing 1 hour prior to stroke and 24 and 48 hours after stroke significantly reduced infarct volume in the cortex by  Oxidative stress contributes to brain reperfusion injury following stroke . Well-esSeveral drugs that target oxidative stress have been developed as potential therapies for ischemic stroke. Spin trap free radical inhibitors and antioxidants, including Ebselen (a glutathione peroxidase mimetic); NXY 059 ; edaravone  can reduce infarct volume in rodent reperfusion models of ischemic stroke, supporting the contribution of reactive oxygen species to ischemic damage following reperfusion [NADPH oxidase (Nox) is the major enzyme source of reactive oxygen species in the vascular system , 7, inflApocynin (4-hydroxy-3-methoxy-acetophenone) is a reported inhibitor of Nox1- and Nox2-dependent superoxide generation in peripheral inflammatory cells  and has All animal experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986, under the guidelines of the National Health and Medical Research Council for the Care and Use of Animals for Experimental Purposes in Australia. The protocol was approved by the St Vincent's Hospital Animal ethics committee (AEC 31/06). All surgery was performed under general anesthesia, and all efforts were made to minimize suffering which included access to paracetomol (2 mg/kg in drinking water) for 24 hours prior to and following surgery, as well as extensive monitoring of each rat throughout the duration of the study. 20 adult male Hooded Wistar rats weighing 300 g to 350 g  were included in this study. Rats were group-housed (4 rats to a cage), until endothelin-1-induced middle cerebral artery constriction, whereupon they were housed in separate cages under diurnal lighting with ambient temperature maintained between 20 and 22°C. Rats were given free access to food and water. Rats were anesthetized with pentobarbitone sodium (60 mg/kg dose i.p.) and a 23-gauge cannula was stereotaxically inserted into the piriform cortex 2 mm dorsal to the middle cerebral artery  as previously described , 16. FolμL saline over 10 min) via a 30-gauge injector needle that protruded 2 mm beyond the end of the previously implanted guide cannula [n = 6 in total) as we have previously shown limited damage after stroke with scores below 3 [Focal cerebral ischemia was induced in the conscious rat by constriction of the right middle cerebral artery with perivascular administration of endothelin-1  (50 mg/kg given i.p.) or vehicle  was delivered 1 hour prior to stroke induction and then again at 24 and 48 hours after reperfusion . The dosing regimen for apocynin was based on previous work by Tang and colleagues  in a ratNeurological abnormalities were evaluated with the use of a neurological deficit score that is based on detection of abnormal posture and hemiplegia described by Yamamoto and colleagues . Brieflyμm) were serially cut in a cryostat  at eight predetermined planes throughout the brain (−3.2 to 6.8 mm relative to Bregma). The extent of brain injury following stroke was determined in triplicate unstained slide mounted sections using a micro computer imaging device  for ballistic light image analysis shown to permit rapid and reliable measurements of cellular damage according to the methods of Callaway and colleagues [Rats were decapitated 72 hours after stroke, forebrains removed, frozen over liquid nitrogen, and stored at −80°C prior to processing. Coronal forebrain sections  was used to evaluate production of ROS in slide mounted sections adjacent to those utilized above for identification of ischemic damage, as previously described and reported by others [μM), for 30 min at 37°C prior to co-incubation with dihydroethidium for 30 min at 37°C. All resulting sections were examined with a fluorescence microscope (Zeiss Axioskop2) equipped with a 590 nm long pass filter set for detection of red fluorescence (dihydroethidium). The oxidative fluorescent indicator dihydroethidium . Images were captured with a computer-controlled digital camera (Zeiss AxioCam MRc5 connected to AxioVision software) using the same fluorescence settings for all rats. The intensity of red fluorescence was calculated by tracing around individual cells and quantitatively analysed in ImageJ software . The ratIn order to identify cell-specific ROS generation frozen sections adjacent to those used in dihydroethidium studies were preincubated for 1 hour in 10% normal Donkey serum prior to overnight incubation with neuronal mouse monoclonal anti NeuN  or mouse monoclonal antibody MRC-II OX-42 for microglia . Tissue sections were then washed in phosphate buffered saline  and transferred for incubation with the secondary antibody, Alexa Fluorophore 488 nm donkey anti-mouse IgG at 1 : 500  in phosphate buffered saline containing 2% normal goat serum and 0.3% Triton X-100 for a further 90 min at room temperature. Sections were then double labelled with dihydroethidium as described above for colocalization of superoxide and immunofluorescence. Sections were cover slipped using fluorescent mounting medium (Dako), and both red and green fluorescence emitted from dihydroethidium and each primary antibody, respectively, were detected using a fluorescent microscope equipped with both a 590 mm and 515–565 mm filter set . Analogous experiments were also conducted with mouse IgG control serum in place of the primary antibody.For detection of Nox2 protein expression sections corresponding to those used above were treated separately with mouse monoclonal anti-gp91Phox  as previously described  and analFor morphological assessment of microglia reactivity, MRC-II OX-42 immunohistochemistry was visualized using cobalt-nickel-enhanced diaminobenzidine according to the protocol described previously . Brieflyn = 6 are required for 80% power where a reduction in infarct by 30% is considered significant with a SD of approximately 15% for each group [P < 0.05 was considered to be statistically significant. Statistical analyses and all graphical representations were generated using Graph Pad Prism 5.01 software.Neurological outcome data was analysed by nonparametric Kruskal-Wallis test. Physiological data and infarct volume was analyzed by two-way ANOVA for comparison between apocynin- and vehicle-treated groups, or for comparison between ipsilateral and contralateral sides following stroke. For analysis of dihydroethidium fluorescence and OX-42 counts within groups following stroke, a one-way repeated measures ANOVA (treatment x h after stroke) was performed. Individual comparisons were made using Tukey's test for all analyses where ANOVA yielded a significant result. We have previously shown that ch group . All datP < 0.05; ANOVA with repeated measures for minutes after stroke), but returned to normal limits after this time (data not shown). This temperature increase at 30 min was less than 1°C for all groups and was, therefore, unlikely to induce nonischemic-related damage.Rats did not lose weight as a result of surgery or stroke as in previous studies . Rectal P < 0.01; Neurological deficit tests were conducted in both treatment groups as a measure of functional outcome following stroke-induced brain injury. Endothelin-1-induced stroke resulted in significant neurological deficits 24 to 72 hours after stroke when compared to prestroke scores in all groups (P < 0.05 two-way ANOVA)   . Infarct; ANOVA) . TreatmeThe superoxide-sensitive indicator dihydroethidium was used to localize and quantitate the effect of apocynin on ROS generation from individual cells following stroke as in previous studies . RegionsP < 0.01; Figures Total dihydroethidium fluorescence within the ipsilateral cortex Figures  was signTo investigate the effect of cell-specific superoxide generation after stroke with apocynin treatment, sections were simultaneously labelled with dihydroethidium and the activated microglia/macrophage marker OX-42 , or dihyP < 0.01 ANOVA; P < 0.001 ANOVA; P < 0.01 ANOVA; Total dihydroethidium fluorescence from OX-42 double labelled cells within the ipsilateral cortex was significantly increased when compared with similar cells from the corresponding contralateral region (n = 15 cells per region). Dihydroethidium fluorescence from individual surviving NeuN positive cells from vehicle-treated rats was significantly reduced in the ipsilateral cortex when compared with similar cells from the corresponding contralateral region .P < 0.001 ANOVA; P < 0.001 ANOVA; In order to determine if reduced Nox2 protein expression in apocynin-treated rats following stroke was associated with changes in inflammatory cell numbers, light field OX-42 immunohistochemistry was utilized to compare the number of activated microglia/macrophages in the infarct core of the ipsilateral cortex where an equivalent area of damage was demonstrated in both vehicle-  and apocin vivo following treatment with apocynin, in particular an unexpected increase in neuronal ROS 3 days after stroke, compared to vehicle controls. This study highlights the need to investigate the mechanisms through which individual cells respond to apocynin and treatments that may target specific NADPH oxidases.Increased levels of reactive oxygen species, in particular superoxide, are a major cause of cellular damage and lesion progression following cerebral ischemia. One of the most important sources of superoxide in mammals is NADPH oxidase . In the Stereotaxic application of endothelin-1 to the middle cerebral artery is now a well established method of focal ischemia in conscious rats  with recAnother consideration for lack of effect in the striatum is the time in which infarct assessment has been conducted. Previous reports assessing the cytoprotective effects of apocynin measured infarcts only up to 24 hours after reperfusion, at times when the infarct may not have fully matured in the models used for assessment –14, 27. in vitro where apocynin attenuated superoxide release from neutrophils and macrophages without effect to inflammatory cell activation [NADPH oxidase and inflammation following stroke has been studied in more detail in recent years for its contribution to the expansion of the core lesion in the days following reperfusion . In the tivation , 31. ColThe mechanism of action through which apocynin attenuates ROS has been the topic of current debate with a report suggesting that apocynin is an antioxidant rather than a specific inhibitor of NADPH oxidase, at least in the vasculature . Others in vivo following stroke. Differential effects on superoxide generation have been observed with apocynin in vitro: Vejražka and colleagues [in vitro, apocynin initially caused an increase in ROS production that was quickly followed by ROS inhibition [The differential response to superoxide in the current study highlights the importance of investigating individual cell responses to superoxide lleagues  also rephibition . This efhibition . In the hibition . DespiteA more likely explanation for increased neuronal superoxide in apocynin-treated rats could be related to the progression of injury over time. We previously reported increases in superoxide generation within activated neurons from the core infarct 6 hours after stroke, an effect that was later observed in the outer penumbra by 24 hrs, and absent by 3 days once the infarct had matured and neurons lost . The detOurs is the first study to examine the effects of apocynin beyond a 24 hr recovery period after stroke and reperfusion. Given that changes in neuronal oxidative stress 3 days after stroke in apocynin-treated rats appear not to be the result of increased NADPH oxidase activity, it is likely that inhibition of Nox2 does not prevent the expansion of the lesion but merely delays its progression. Studies have now demonstrated that treatments targeting oxidative stress and inflammation should be assessed over longer time periods because although a compound may be initially effective with a short survival time, beneficial effects may be lost with longer periods of recovery . Indeed Using a rat model of conscious stroke, we report for the first time that apocynin treatment, prior to and after stroke, reduces inflammatory cell-mediated superoxide generation through attenuation of increased Nox2 expression that occurs in response to stroke-induced brain injury. Additionally, we show that neurons in the cortex of apocynin-treated rats are in a state of oxidative stress indicative of progression of injury suggesting that targeting Nox2 after stroke merely delays the progression of brain injury rather than prevent it. The outcomes of this study in comparison to previous work highlight the importance of preclinical testing of potential cytoprotective agents in multiple species, in more than one model of stroke, but most importantly with inclusion of extended recovery times that mimic more closely the human scenario.


Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.The  The molecular mechanisms that allow painless to have polymodal sensory roles that include thermosensory, chemosensory, and mechanical signaling are not yet understood.Transient receptor potential (TRP) channels are important in the senses of vision, taste, touch, hearing, nociception , and thermosensation  behavior that is seen in larvae exposed to noxious heat painless mutant larvae show increased sensory thresholds for nocifensive responses to noxious heat as well as responses to noxious mechanical stimuli. Evidence suggests that the Painless channel is a direct sensor of noxious heat. For example, electrophysiological recordings from painless mutant larval abdominal thermosensory neurons showed decreased firing in response to noxious temperatures in vitro heat activation threshold for Painless is similar to the 39–41°C behavioral threshold for triggering larval NEL. The nociceptive function for Painless is likely mediated by nociceptive Class IV multidendritic (mdIV) neurons which express the gene and also show strong increases in firing at temperatures >39–41°C pickpocket gene is specifically expressed in the mdIV neurons and it is required for mechanical nociception The nociception function of painless mutant animals relative to wild type larvae which indicates an in vivo requirement for Painless in mechanical nociception responses in vitro studies on Painless expressed in a human embryonic kidney cell line failed to detect Painless dependent currents following hypo/hypertonic stimulation or direct touch with a glass pipette Drosophila larvae but it is not required for thermal nociception 2+ which may provide a potential molecular mechanism for activation downstream of neuronal activity With regard to mechanical nociception, the role of Painless is more poorly understood. The NEL responses to noxious mechanical stimulation have an increased threshold in C. elegans, the mechanotransduction complex relies on the pore forming DEG/ENaCs MEC-4 and MEC-10, extracellular proteins  and additional intracellular components Drosophila, the extracellular protein NompA, is required for the connection between the mechanosensory neuronal sensory endings and cuticle in bristle mechanotransduction On the other hand, heterologous expression studies are difficult to interpret since mechanosensative channels may require specialized signaling components that may not be present in heterologous cells painless that are predicted to encode Painless protein isoforms which vary in the length of the ankyrin repeat containing N-terminal domain. We utilize these naturally occurring variants as tools to investigate the functional properties of the ankyrin repeat domain of Painless through isoform specific rescue experiments in vivo. Our results indicate that the longest isoform, which contains the entire N-terminal ankyrin repeat domain, is sufficient to rescue thermal nociception of a painless mutant but does not rescue mechanical nociception. In addition, this long protein isoform shows efficient localization to dendrites and axons of the multidendritic neurons. In contrast, the shortest isoform, which essentially lacks an ankyrin repeat domain, is capable of rescuing mechanical nociception without rescuing thermal nociception. This short isoform does not efficiently localize to dendrites and is expressed at much lower levels in comparison to the long isoform. Combined, these results suggest that the polymodal functions of Painless may be explained by distinct protein variants that have modality specific properties. Furthermore, our results suggest that the N-terminal domain of Painless is required for thermal nociception, but we do not find evidence that it is required for mechanical nociception.Given the polymodal role of Painless in thermal nociception and in mechanical nociception we were interested in the possibility that particular domains of the Painless protein might play an important role in various aspects of signaling. Here, we report three naturally occurring RNA variants of painless are detectable by northern blot analysis painless, we performed 5′ Rapid Amplification of Complementary cDNA Ends (5′ RACE). Three 5′ RACE products were cloned and sequenced and determined to represent three distinct 5′ ends for painless transcripts. One of the RACE products was identical to the previously described 5′end of painless while the remaining two RACE products encoded novel 5′ ends. A search of the expressed sequence tag (EST) database identified 5′ EST sequences that exactly matched the 5′ ends of the painless RACE products. Only a single 3′EST sequence for painless transcripts is found in the database and this 3′end is identical to that of the canonical painless transcript. Combined, these data are consistent with the existence of three painless transcripts that differ in the 5′ sequences but which share a common 3′end. Conceptual translation of the novel transcripts predicted proteins that differed in the translational start site of their N-terminal domains. Based on the predicted molecular weights of the isoforms , we refer to the predicted protein isoforms as Painlessp103, Painlessp72, and Painlessp60.Three transcripts of p103, is predicted to have 8 N-terminal ankyrin repeats, 6 transmembrane domains, and an intracellular carboxy-terminal domain (CTD) p103. The transcript encoding the intermediate length isoform, Painlessp72, is transcribed from the same promoter as the transcript encoding Painlessp103, but it includes an alternately spliced second intron that results in the utilization of an alternate ATG start codon  isoform, Painlessrt codon . The resnscripts . This stin vivo, we generated transgenic files to specifically express either the Painlessp103 isoform or the Painlessp60 isoform under control of the GAL4/UAS system. In order to estimate expression levels of the transgenes, an in-frame fusion of the Venus Fluorescent Protein (VFP) was added at the C-terminus of both constructs. We refer to these transgenes as p103::VFPUAS-painless and p60::VFPUAS-painless. To examine the localization of the VFP tagged Painless proteins, we crossed flies harboring p103::VFPUAS-painless or p60::VFPUAS-painless to the GAL4painless driver strain. In the case of p103::VFPUAS- painless the progeny of this cross showed robust VFP fluorescence throughout the dendrites, cell bodies, and axons of the GAL4painless expressing multidendritic sensory neurons  of the transcript encoding for Painlessp103 and the shared 5′ UTR of the transcript encoding Painlessp72. The Gal4pain allele shows GAL4 expression in multidendritic neurons, chordotonal neurons, and a subset of neurons in the CNS In order to study the potentially distinct roles of the PainlessGal4pain mutant allele in combination with the NP7022pain allele. Mutant animals with the Gal4pain/NP7022pain genotype showed pronounced thermal nociception and mechanical nociception defective phenotypes. In wild type larvae gently touched with a probe heated to a noxious temperature (46°C), nocifensive escape behavior was rapidly triggered  showed a nearly complete rescue of nociception responses to a 46°C thermal stimulus  generates transcripts that encode either heat sensitive  or heat insensitive isoforms of dTRPA1  dTrpA1 could rely on an as yet unidentified isoform for this channel that lacks ankyrin repeats as we have now found for painless.Interestingly, recent results from our laboratory Drosophila TRPA1 channels made with the heat insensitive human TRPA1 channel, support the idea that heat sensor domains reside within the ankyrin repeat region in vivo analyses of painless are in definite support of this idea.In addition, chimeric channels between heat sensitive snake or 2+ influx, since the putative EF hand of the Painless NTD in absent in the Painlessp60 isoform.Although the Painless NTD is apparently not required for mechanical nociception it would be premature to extrapolate from this finding to other TRP channels involved in mechanotransduction. Additional experiments will be necessary to test whether or not Painless or other TRPA channels encode pore-forming subunits of mechanosensory channels. As mentioned above, Painless may function downstream of another mechanosensory such as Pickpocket. Interestingly, our results suggest that if Painless does function downstream of Pickpocket, then the method of activation for Painless is unlikely to be dependent on Cap60::VFP transgene was expressed at relatively low levels in vivo and its subcellular localization was restricted to proximal dendrites, the cell soma, and proximal axons. In contrast, the Painlessp103::VFP isoform was expressed robustly and throughout the multidendritic neurons including the sensory dendrites. These findings suggest the possibility that Painlessp103::VFP and Painlessp60::VFP proteins function in distinct subcellular compartments for thermal and mechanical nociception. Thermal nociception signaling by Painless p103 may occur in the more distal sensory dendrites whereas the mechanical nociception function for Painless may reside in proximal regions of dendrites, likely in amplification of upstream mechanosensory transduction signals initiated by the DEG/ENaC Pickpocket.Our results also indicate that the Painless ankyrin repeats play an important role in the sub-cellular localization and the expression level of Painless isoforms. The Painlesspainless transcripts were isolated by 5′ RACE using the FirstChoice  according to the instructions of the manufacturer. RNA was isolated from a mixed population of first and second instar 1118Drosophila w larvae using Trizol reagent (Invitrogen/Life Technologies) and treated with DNAse-I (DNA-free Kit (Ambion Inc.). The 5′ phosphate from any uncapped RNA in the preparation was then removed by treatment with Calf Intestine Alkaline Phosphatase (CIP). The CIP reaction was terminated by phenol-chloroform extraction, and the RNA was precipitated with isopropanol. The 5′ cap was removed from the mRNA by treatment with Tobacco Alkaline Pyrophosphatase (TAP) enzyme, leaving monophosphated 5′ ends. A 45-base RNA adaptor from the FirstChoice  and are complimentary to sequences present in the ligated 5′ adaptor. The outer reaction painless specific reverse primer 5′-GGATGGTAAATACGGCTAAGAC-3′ and the inner reaction painless-specific reverse primer 5′-TTCGTGGAACTTGAGGAGGCGTG-3′ were used. PCR products were examined by agarose-TAE gel electrophoresis. The products were gel-purified, cloned into pCR-XL-TOPO cloning vector (Invitrogen/Life Technologies), and introduced into electrocompetent E.coli of the TOP10 strain (Invitrogen/Life Technologies). DNA from Kanamycin-resistant colonies was digested with EcoRI restriction endonuclease to release the cloned inserts, and the relative molecular weight of the inserts was determined by gel electrophoresis. Clones containing inserts with unique molecular weights were selected for sequencing. The inserts of clones were sequenced using M13 F and M13R against the TOPO XL vector.First strand cDNA was generated using random decamer primers and M-MLV Reverse Transcriptase. Nested PCR was performed to amplify p103::YFPpainless and UAS-p60::YFPpainless plasmids the open reading frames were amplified from BACR08I14 using the forward primer for p103painless5′ CAC CAT GGA CTT TAA CAA CTG C ′3 or the forward primer for p60painless 5′ CACCATG GAT ATC AAC TCG AGA CCA 3′. The reverse primer 5′ CCG GTC CTG GAC CAG CT 3′ was used for both constructs. The PCR products for the respective gene products were then cloned in the pENTR-D vector (Invitrogen) for use with the Drosophila Gateway Cloning System. The resulting p60painless  and p103painless ENTR vectors were then used as substrates for clonase mediated recombination with the pTWV destination vector. Fully sequenced inserts from this reaction were found to contain wild type painless sequences and were used to transform the 1118 Drosophilaw strain via P-element mediated transformation. Inserts from transformed flies were mapped to a chromosome and the expression levels of the VFP transgenes were determined by crossing to the GAL4painless driver strain. For behavioral experiments inserts on the third chromosome were used. Flies with the genotype GAL4w;painless, GAL4w; painless; UAS-p103::YFP/painlessK87 (T(2∶3) SM6a TM6b Cy Tb), or GAL4painless; UAS-p60::YFP/painlessK87 were crossed to NP7022/painK87 and +Tb progeny were selected and tested for nociception behavioral responses as described previously.To generate the UAS-GAL4pain driver was crossed P103::VFPto UAS-painless (venus fluorescent protein) or P60::VFPUAS-painless. Trans-heterozygous larvae were dissected and filleted in Ca2+ free HL3 saline  followed by fixation for 1 hour in 4% paraformaldehyde. Primary mouse anti-GFP (1∶1000) and anti-mouse Alexa Fluor 488  secondary were used to detect VFP. Neurons were counterstained with rabbit anti-HRP  and the secondary anti-rabbit Alexa Fluor 568 . Images (1024×1024) were taken with a Zeiss LSM 5 Live Confocal system using a 40×1.3 N/A oil immersion lens. The two channels were collected separately in multi-track mode  . The confocal micrographs are presented maximum intensity projections of confocal Z-stacks.For visualization of the painless isoforms, the


Chytridiomycota as well as occurrences of Cryptomycota and yeast-like fungi confirm the decomposer niche overlap of saprotrophic fungi and bacteria on pollen. As hypothesized, microbial communities consistently differed between the lake types and exhibited functional differences. Bacteria-to-fungi ratios correlated well with parameters such as organic carbon and pH. The importance of dissolved organic carbon and nitrogen for bacteria-to-fungi ratios was supported by the Baltic Sea river dataset. Our findings highlight the fact that carbon-to-nitrogen ratios may also control fungal contributions to OM decomposition in aquatic ecosystems.Fungi and bacteria are the major organic matter (OM) decomposers in aquatic ecosystems. While bacteria are regarded as primary mineralizers in the pelagic zone of lakes and oceans, fungi dominate OM decomposition in streams and wetlands. Recent findings indicate that fungal communities are also active in lakes, but little is known about their diversity and interactions with bacteria. Therefore, the decomposer niche overlap of saprotrophic fungi and bacteria was studied on pollen  by performing microcosm experiments with three different lake types. Special emphasis was placed on analysis of fungal community composition and diversity. We hypothesized that (I) pollen select for small saprotrophic fungi and at the same time for typical particle-associated bacteria; (II) fungal communities form specific free-living and attached sub-communities in each lake type; (III) the ratio between fungi or bacteria on pollen is controlled by the lake's chemistry. Bacteria-to-fungi ratios were determined by quantitative PCR (qPCR), and bacterial and fungal diversity were studied by clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprints. A protease assay was used to identify functional differences between treatments. For generalization, systematic differences in bacteria-to-fungi ratios were analyzed with a dataset from the nearby Baltic Sea rivers. High abundances of  The functional role of aquatic fungi and bacteria in decomposing vascular plant litter in streams and marshlands has been assessed frequently , and establishment of hyphal networks might be retarded due to their limited surface size. It has been already shown that the fungal biomass on POM in streams measured by ergosterol decreased with decreasing POM size class, which suggests a bacterial predominance on smaller particles 2 = 0.91, Saprotrophic fungi and saprotrophic bacteria seem to have a pronounced niche overlap, and both depend strongly on the organic carbon content across biomes  was used as a metric to characterize soil ecosystems To test for the importance of aquatic fungi on small FPOM, we performed a laboratory microcosm experiment with pollen grains and studied their diversity, community structure, and the decomposer niche overlap with bacteria on pollen using water from three lake types differing in refractory DOC and pH. We hypothesized:that small unicellular and zoosporic aquatic fungi will greatly benefit from input of pollen, in particular saprotrophic fungi;that lake-specific communities of microbes are well adapted to pollen following a progressive succession;that different lake properties define systematic differences between the dominance of fungi and bacteria and therefore shape the niche overlap between both saprophytic microbial groups.P. sylvestris was collected during the pollen season on a dry and canopied surface close to the shore of Lake Stechlin in late May 2008. The water samples were taken at 16th of June 2008, which was also the start of the experiment. Sampled lakes are part of the nature conservation park “Stechlin” in Northeast Germany . Briefly, 100 µm gauze pre-filtered water samples were obtained from oligotrophic Lake Stechlin  and from the eutrophic northeast basin  and the dystrophic southwest basin  of Lake Grosse Fuchskuhle. These two basins are considered as two lake systems with distinct microbial communities −1 of 2–3 weeks old pollen of Pinus sylvestris (freshness/viability was confirmed in the microscope by observing the formation of pollen tubes: 21% of pollen grains in NE water developed tubes) in a total volume of 2 liters  were described in detail by 2 liters . SamplesAll samples were subdivided to measure side variables (120 ml): total organic carbon (TOC), DOC, particulate organic carbon (POC), soluble reactive phosphorus (SRP), total dissolved phosphorus (TDP), dissolved organic phosphorus (DOP), particulate phosphorous (PP), particulate nitrogen (PN), and total dissolved nitrogen . The humic-matter-rich water from SW had a weak quenching effect, which we adjusted for by calibrating each treatment with MCA (Sigma).We exemplarily measured the leucine-aminopeptidase activity at day 3 when bacteria were dominant. Samples that had been incubated for three days without any pollen addition served as controls. Fluorogenic leucine-MCA (Sigma) was added at saturating concentration (0.1 mM) to a 4 ml subsample as a substrate analogue. After 1 h of incubation at 18°C, pH was adjusted to pH>10 using borate buffer directly before fluorescence measurements −1, 2 min). DNA concentrations were measured via PicoGreen (Invitrogen) using a microplate reader .Stored filters were extracted with a peqGOLD Tissue DNA Kit  with an additional upstream mechanical disruption step in which the filters were added to 0.25 g of 0.1 mm zirconium beads and mechanically disrupted by bead beating in a benchtop mill  modified from LR7 AAGCATATCAATAAGCGGAGG), modified from CCTTAGACTGACAGATTAA) and QRTfungr (GTTTGATCTCAGTTCGAG). Detailed information on primer design, evaluation, regions, and handling is given in the supporting information (Microscopic observations  confirmeormation .2) with the following cycling conditions for fungal-specific primer pair LR78/RD78: 33 cycles of 95°C for 1 min and 2.5 min at 60°C annealing/elongation. Fungal PCR products were subsequently separated in a denaturing gradient of 30–45%. For bacterial DGGEs, we utilized primer pair 341f-GC and 907r with DGGE conditions as described in DGGE was performed following Escherichia coli. To preselect clones for sequencing, we screened at least 96 clones per library and digested the insert with the enzymes MSPI and RsaI . The obtained banding pattern was analyzed on a 3% agarose gel stained with SybrGold . Representative clones of inserts with potentially different restriction fragment length polymorphism (RFLP) patterns were selected and their inserts were sequenced with primers T7, SP6, and NL4Seq (TTGAAACACGGACCAAGGA). Two additional clone libraries were prepared to compare DNA for both size fractions (PA and FL) without pre-selection. For this, we used DNA from the ST treatment at day 5.Clone libraries were constructed solely for fungi. Approximately 2000 nt of the LSU rRNA gene were amplified, spanning regions D1 to D8, using primer NL1a and RD78 with the following cycling conditions: 95°C for 3 min; 35 cycles at 95°C for 1 min and 60°C for 4 min; and a final elongation step for 30 min at 68°C. Obtained PCR products were quality checked and purified with silica columns (Qiagen PCR purification). For phylogenetic comparison of lake types (treatments), PCR products derived from the DNA of both size fractions were pooled together. Similarly, all those pools derived from the two early sampling dates (days 2 and 5), and the two from the late sampling dates (days 10 and 16) of one replicate per treatment were merged, which resulted in six pools. These were cloned into pGEM-T Easy (Promega) vectors and transformed in All full-length sequences of our clone libraries (>1.8 knt) were aligned using the SINA web aligner V1.2.9;  and inteIn order to investigate the biomass-dynamics of fungi and bacteria during pollen degradation, we followed molecular quantities of ribosomal gene copies. For qPCR, we used EvaGreen (BioRad) with a 25–500 pg template in a total volume of 13 µl; a CFX96 cycler (BioRad); our newly designed primer pair QRTfungf and QRTfungr to quantify fungal ribosomal copy numbers ; and primer pair Ba519f and Ba907r for ribosomal bacterial genes (under conditions according to http://www.r-project.org/). Significant category separations  were investigated at a 0.95 confidence level and were visualized by plotting the deviation from average using an ellipse based around centroids. To account for possible metric factors that influence the community structure presented in the NMDS, chemical parameters and time were fitted into the two-dimensional ordination as vectors . The relationships between bacterial and fungal fingerprints were examined using a Mantel test DGGE community profiles were transformed into a digital binary matrix with the GelCompar II software package . Internal variation among replicates was resolved by cluster analysis based on Dice dissimilarities and UPGMA. This internal control assured sufficient similarity between the replicates  and allowed choosing a representative sample for time-series analysis . For funFor qPCR data, a two-way analysis of variance (ANOVA) with lake type and time as well as size fraction and time as factors was applied. Data were ln-transformed if needed (Smirnov-Kolmogorov test), and Levene's test was used to ensure the homogeneity of variances. Similarly, differences in aminopeptidase activities were confirmed using one-way ANOVA with Tukey's HSD-Post-Hoc test (α<0.05).−1. Based on the assumption that a combination of environmental variables determines B∶F, we further computed the least absolute shrinkage and the selection operator (LASSO) with the least angle regression solution for possible predictors using the R package “lars” (http://cran.r-project.org/web/packages/lars/index.html) to select the most probable predictor 2 = 0.152, dF = 32) vs. null model: p = 0.024; DOC+DON vs. DOC-model: p = 0.038).The differences in average B∶F ratios between treatments were analyzed by a non-parametric Kruskal-Wallis test . Furthermore, the late phase of the experiment (approximating the climax community) allowed us to set the B∶F ratio in relation to external factors. Since we were operating in a closed system, we expected that the B∶F would in turn influence the dissolved parameters. Hence, we chose to calculate the Spearman's rank correlation between the two measures. In order to investigate a possible linearity with C∶N we ln-transformed the data and calculated the Pearson's correlation. In a continuative step and as a conclusion of the results see the  section,Pollen rapidly decomposed within 16 days of incubation. After pollen were exposed to lake water, pollen delivered an easily accessible soluble and particulate OM. Zoosporic fungi and filamentous forms emerged, which led to an aggregation of pollen grains Cryptomycota were likely underestimated by qPCR due to a primer mismatch selecting against them and therefore might be of larger importance in Lake Stechlin than seen in the qPCR. The pronounced decline of both microbial groups at the end of the experiment might mark the beginning of the death phase due to starvation (pollen grains were completely emptied at that time), grazing, parasitism, viral lysis, or accumulation of toxic metabolites.Our qPCR data confirm earlier observations: free-living bacteria exhibited an early maximum, whereas fungi appeared after a lag phase (estimated then by number of sporangia per pollen grain) Dikarya, we were successful in spanning a broad evolutionary distance across fungal lineages  and 8 sequences to three fungal-like groups . Chytridiomycota sequences were prevalent (49%), and most of them were clustered into the order Rhizophydiales. They were followed by a group of sequences in proximity to Rozella sp. (30%), which we affiliated to Cryptomycota (Dikarya); most of these sequences belonged to the dimorphic genus Filobasidiella (Basidiomycota). The remaining 8% of the sequences fell within the fungal-like phyla, and Oomycetes was prominent.Our microscopy studies revealed a variety of basal forms as well as filaments and propagules (conidia) from higher fungi . We aimelineages . Howevertomycota . These sRhizophydiales). Several lake studies demonstrated that this order includes a number of freshwater lineages without any close relatives RhizophydialesCryptococcus species (yeast form of Filobasidiella species) can grow dimorphically . Cryptomycota were represented by 30 clones, and 93.3% of them belonged to the FL fraction (Chytridiomycota (84.6% were PA), they should demonstrate a similar apportionment. These results clearly show a free-living (possibly yeast-like) saprotrophic life for the retrieved Cryptomycota cluster from Lake Stechlin. Basidiomycota were also detected in the FL fraction which may point to a yeast-like growth, however, they were only represented by a single clone.Furthermore, we studied the fungal phyla distribution between the three lake systems and their occurrences during the incubation period . With relineages . During periment . The lowms e.g., . Cryptomfraction . If theyBetaproteobacteria to be most prevalent (53%); this bacteria frequently colonizes organic aggregates such as lake snow Betaproteobacteria are among the most widely distributed phyla in freshwater systems Betaproteobacteria are known to grow preferentially on polymeric organic matter Actinobacteria (12%), Alphaproteobacteria (9%), Bacteroidetes (5%), Firmicutes (2%), and Cyanobacteria (2%). The retrieved chloroplast sequences (17%) belonged to Chrysophyceae and Pinus sylvestris (added pollen). We concluded that Betaproteobacteria and Chytridiomycota were the most successful in our pollen incubations.In contrast to fungal organisms, we roughly delineated the occurring bacteria by obtaining 43 sequences derived from DGGE bands (data not shown). We found Cryptomycota pointed to a strong difference in fungal communities introduced by habitat water from the different lakes. DGGE analysis revealed a precise overview on the temporal dynamics of both microorganism groups during the incubation. NMDS analyses of the DGGE fingerprints from fungi and bacteria clearly reflected the different lake types  to coexist with bacteria on and surrounding pollen particles. Fungal and bacterial saprobes are enriched to a different extent, and this seems to rely greatly on specific environmental factors. Fungi were most prominent in acidic dystrophic water, which represents a highly abundant lake type in temperate and boreal regions. In those regions, pollen input can be substantial. In such lakes, fungi would contribute to a substantial part of the microbial loop with important ecological implications, e.g., by altering the entire food-web by adding cross-trophic links to zooplankton filter-feeders The high biodiversity of co-occurring heterotrophic microorganisms on allochthonous pollen is remarkable since pollen represents a rather uniform, carbon- and nutrient-rich, microbial substrate. Particles produce an additional spatial arrangement that allows for more ecological niches than simple solutes. In particular, pollen favors a diverse fungal community  and day 16 (Lake-16). 0.2 marks the free-living and 5.0 marks the particle-associated size fraction. Cluster analysis is based on the presence/absence of DGGE bands (Dice) and was calculated with average distances (UPGMA).(TIF)Click here for additional data file.Figure S2Leucine-aminopeptidase activity at day 3. Leucine-aminopeptidase activity of the three treatments after pollen addition. Controls consist of treatment water incubated without pollen for three days, respectively.(TIF)Click here for additional data file.Figure S3Global bacterial versus fungal variation in copy numbers. Absolute copy numbers of all data points show no global trend between bacteria and fungi. Size fractions were added.(TIF)Click here for additional data file.Figure S4LASSO analysis of B∶F ratio calculated from the Baltic Sea catchment dataset. LASSO of multiple standardized variables that are most deterministic for the B∶F ratio in 33 rivers of the Baltic Sea .(TIF)Click here for additional data file.Table S1Chemical parameters.(TSV)Click here for additional data file.Material S1Primer evaluation.(DOC)Click here for additional data file.Material S2Short and partial sequences.(TSV)Click here for additional data file.


Bacillus subtilis and Aspergillus oryzae for 6 days modulated insulin resistance, insulin secretion, and pancreatic β-cell growth and survival in 90% pancreatectomized (Px) diabetic rats, a moderate and non-obese type 2 diabetic animal model.Although soybeans have the ability to attenuate insulin resistance, it is insufficient to alleviate type 2 diabetic symptoms and different types of fermented soybeans may have even better anti-diabetic effects. Meju, unsalted fermented soybeans exhibited better insulin sensitizing and insulinotropic actions than unfermented cooked soybeans (CSB). We investigated whether meju fermented in the traditional (TMS) manner for 60 days and meju fermented in the standardized (MMS) method inoculating Diabetic rats were divided into 3 groups: 1) TMS (n = 20), 2) MMS (n = 20) or 3) casein . Rats were provided with a high fat diet (40 energy % fat) containing assigned 10% meju for 8 weeks. At the end of experiment insulin resistance and insulin secretion capacity were measured by euglycemic hyperinsulinemic clamp and by hyperglycemic clamp, respectively. Additionally, β-cell mass and islet morphohometry were determined by immunohistochemistry and insulin signaling in the liver was measured by western blot.TMS and MMS increased isoflavonoid aglycones much more than CSB. CSB and TMS/MMS improved glucose tolerance in diabetic rats but the mechanism was different between treatments (P < 0.05). CSB enhanced peripheral insulin sensitivity including hepatic insulin sensitivity better than the control but TMS and MMS enhanced only hepatic insulin sensitivity through activating insulin signaling in diabetic rats (P < 0.05). However, TMS and MMS, but not CSB, potentiated glucose-stimulated insulin secretion and β-cell mass (P < 0.05). MMS had better insulinotropic actions than the control (P < 0.05).Bacillus subtilis and Aspergillus oryzae, was superior to CSB by increasing isoflavonoid aglycones and small peptides with regard to type 2 diabetic rats.The anti-diabetic action of MMS, especially when fermented with  Glucose homeostasis is maintained by the hyperbolic relationship of insulin sensitivity and insulin secretion. When insulin secretion can compensate for insulin resistance, normoglycemia can be maintained. Type 2 Glycine max MERILL) have been consumed as an important protein source to complement grain protein in Asian countries over a long period of time. Besides soy protein, they contain various nutritious and functional components such as isoflavonoids, which are helpful in protecting against metabolic diseases such as obesity and type 2 diabetes glucose  was continuously infused during a four-hour period at the rate of 0.05 μCi/min. Basal hepatic glucose output was measured in blood collected at 100 and 120 minutes after initiation of the [3-3H] glucose infusion. A primed continuous infusion of human regular insulin  was then initiated at a rate of 20 pmol kg–1 min–1 to raise plasma insulin concentration to approximately 1100 pM after 210–240 min. Blood samples from arteries were collected at 10-minute intervals for glucose evaluation, and 25% glucose was infused as needed to clamp glucose levels at approximately 6 mM. Disintegrations per min (dpm) of plasma [3-3H]-glucose with and without drying were measured; plasma concentration of 3H2O was determined by the difference between 3H counts with and without drying. Rates of whole body glucose uptake and basal glucose turnover were determined according to the ratio of the [3-3H] glucose infusion rate to the specific activity of plasma glucose (dpm/mmol) during the final 30 minutes. Hepatic glucose production at the hyperinsulinemic clamped state was determined by subtracting the glucose infusion rate from the whole body glucose uptake. After clamp, the rats were immediately anesthetised with a mixture of ketamine and xylazine and were killed by decapitation. Tissues were rapidly collected, weighed, frozen in liquid nitrogen, and stored at −70°C for further experiments.After catheterisation of the right carotid artery and left jugular vein in the 7th week, a euglycemic hyperinsulinemic clamp was performed on fasted conscious rats (n = 10) to determine insulin resistance as previously described. [3-3H] After seven weeks of treatment, catheters were surgically implanted into the right carotid artery and left jugular vein of ten conscious and overnight fasted rats from each group after anesthetization with ketamine and xylazine . After 5–6 days of implantation, a hyperglycemic clamp was performed in free-moving and overnight fasted rats to determine insulin secretion capacity as described in previous studies. During ser478, Akt, phospho-AMPKthr172, AMPK , and phosphoenolpyruvate carboxykinase (PEPCK), generously provided by Dr. Granner of Vanderbilt University. The primary antibody was diluted with 1000X and secondary antibody was diluted with 5000X. The intensity of protein expression was determined using Imagequant TL .The livers taken from four rats after hyperglycemic clamp were used for an immunoblotting assay. The froAt the end of the 8-week experimental period, nine to ten rats from each group were injected with BrdU (100 μg/kg body weight). Six hours post-injection, rats were anesthetized with intraperitoneal injections of mixture of ketamine and xylazine, and the pancreas was immediately dissected. The pancreas was fixed with 4% paraformaldehyde and paraffin-embedded, as described in previous studies. Two ser+ nuclei in β-cell nuclei per pancreas section while apoptosis of β-cells was measured by the total number of apoptotic bodies in β-cell nuclei per pancreas section[The pancreatic β-cell area was measured by examining all of the non-overlapping images in two insulin-stained sections of each rat at a magnification of 10x with a Zeiss Axiovert microscope . The results of β-cell quantification were expressed as the percentage of the total surveyed area containing insulin-positive cells, measured by IP Lab Spectrum software . Pancreatic β-cell mass was calculated by multiplying the percentage of insulin-positive area by the weight of the corresponding pancreatic portion,12. The  section,12.P < 0.05. The differences between the Px diabetic rats (control) and Sham non-diabetic rats  were determined by two-sample t test.Statistical analysis was performed using SAS software and all results expressed as mean ± standard deviation. The biological and metabolic effects of casein (control), CSB, TMS, and MMS were compared by one-way ANOVA. Pearson’s correlation coefficients were determined between isoflavonoid aglycone contents the rats in the diet and some parameters. Significant differences in the main effects among the groups were identified by Tukey’s test at Bacillus subtilis and Asperilus oryzae. Peptide contents were reduced by fermentation and the reduction was greater in TMS than MMS  and then declined to a nadir at 10 min and then increased and remained steady at 60–90 min (second phase) during hyperglycemic clamp. The serum insulin levels of Px rats were half those of Sham rats during first and second phase insulin secretion under hyperglycemic clamp (data not shown). MMS and TMS increased serum insulin levels during both first and second phase more than the control during hyperglycemic clamp Figure. First aGlucose infusion rates during hyperglycemic clamp indicated β-cell function and insulin sensitivity at hyperglycemic state, calculated as the ratio of glucose infusion rate to steady-state serum insulin levels. GlucoseThe percentage of the pancreatic β-cell area was greater in Px rats than Sham rats as islets were replenished in greater numbers than had existed in the original state when the pancreas was regenerated by up to 50–60% in comparison to the original pancreas as a result of increased proliferation and neogenesis after 90 % of the pancreas had been removed. However, pancreatic β-cell mass, calculated by multiplying β-cell area by the pancreas weight, was greater in Sham rats than Px rats since the pancreas size of Px rats was 50–60% of the Sham rats. Pancreatic weight was significantly higher in Sham rats than Px rats by about 2 folds while it was not significantly different among the groups of Px rats (data not shown). Only MMS increased the percentage of β-cell area more than the control. Although pancreas weight was not significantly different among the differently treated Px rats, total β-cell mass was higher among MMS treated Px rats in comparison to the other groups of Px rats  in cell-based studies[Koreans exhibited a lower prevalence of diabetes but the prevalence has been remarkably increased in recent years. This ma studies. In addiBacillus species[It has been well documented in previous studies that the fermentation of soybeans increases isoflavone aglycones and that the aglycone forms have better bioavailability and functionality due to their enhanced absorption in humans and animals,19. Alth species,21. The  species. These cThe potentiation of β-cell function and mass as a result of increasing proliferation and decreasing apoptosis may be related to increased isoflavonoid aglycones such as daidzein, genistein and glycitein in TMS and MMS in comparison to CSB. Previous studies have revealed that up to 20 μM genisitein improved glucose-stimulated insulin secretion in islets with β-cell proliferation and in insulinoma cells by augmenting cyclic adenosine 3′5′-monophosphate (cAMP) accumulation to activate PKA signaling in a dose dependent manner-25. Howe473 phosphporylation of Akt. Thus, meju, long-term fermented soybeans, demonstrated that it could induce an improvement in hepatic insulin resistance in diabetic rats.Previous studies have reported that soy protein produces lower fasting plasma glucose and insulin concentrations than a casein diet in non-diabetic animal and human studies but recent studies have shown that soybeans do not have a beneficial effect on glycemic control in diabetic humans-31. In tThe contents of isoflavonoid aglycones increased in an ascending order of CSB, TMS, and MMS. The isoflavonoid aglycones contents of MMS were much higher than CSB and TMS—by 29.3 and 2.5 folds, respectively. CBS, TMS and MMS improved glucose tolerance in diabetic rats while CSB did not enhance it as much as MMS and TMS. However, the mechanism of the improvement was different in CSB and MMS/TMS. CSB enhanced peripheral insulin sensitivity including hepatic insulin sensitivity better than the control but TMS and MMS enhanced only hepatic insulin sensitivity in diabetic rats. However, CSB did not improve glucose-stimulated insulin secretion and β-cell mass in diabetic rats while TMS and MMS did potentiate insulin secretion and β-cell mass. In addition, MMS had better insulinotropic actions than TMS. The results of our previous cell-based studies were not exactly consistent with those of the present animal study. The results suggested that MMS and TMS improved glycemic control by potentiating insulinotropic actions and alleviating hepatic insulin resistance in diabetic rats. MMS may be a good candidate as a functional food for relieving diabetes and also postmenopausal symptoms since it contains greater quantities of isoflavonoid aglycones, especially daidzein and genistein.The authors declare that they no competing interests.HJY helped prepare meju and performed all statistical analysis of results; DYK performed to prepare different kinds of meju and to analyze their composition and helped draft the manuscript; MJK helped analyze the contents of isoflavonoids and peptides; ESH helped perform animal study; DSK performed all animal surgery and animal study; SMP overlooked all aspects of the projects, wrote the draft manuscript abd obtained the funding for the work. All authors read and approved the final manuscript.


Plasmodium infection and malaria outcomes. Further studies should include a larger number of individuals, refined parameters and a fine-scale map obtained through DNA sequencing to increase the knowledge of the Amazonian population genetic diversity.The Brazilian Amazon is a hypo-endemic malaria region with nearly 300,000 cases each year. A variety of genetic polymorphisms, particularly in erythrocyte receptors and immune response related genes, have been described to be associated with susceptibility and resistance to malaria. In order to identify polymorphisms that might be associated with malaria clinical outcomes in a Brazilian Amazonian population, sixty-four human single nucleotide polymorphisms in 37 genes were analyzed using a Sequenom massARRAY iPLEX platform. A total of 648 individuals from two malaria endemic areas were studied, including 535 malaria cases  and 113 health controls with no history of malaria. The data revealed significant associations (p<0.003) between one SNP in the IL10 gene (rs1800896) and one SNP in the TLR4 gene (rs4986790) with reduced risk for clinical malaria, one SNP in the IRF1 gene (rs2706384) with increased risk for clinical malaria, one SNP in the LTA gene (rs909253) with protection from clinical malaria and one SNP in the TNF gene (RS1800750) associated with susceptibility to clinical malaria. Also, a new association was found between a SNP in the CTL4 gene (rs2242665), located at the major histocompatibility complex III region, and reduced risk for clinical malaria. This study represents the first association study from an Amazonian population involving a large number of host genetic polymorphisms with susceptibility or resistance to  Malaria is a life-threatening parasitic disease transmitted by mosquitoes. Despite major efforts aimed at controlling the spread and impact of the disease, it still persists as a major health burden, being responsible for over a million deaths each year, mainly children in Sub-Saharan Africa. In Brazil, there were over 300,000 recorded cases of malaria in 2010, almost exclusively (99.8% of the cases) restricted to the Amazon Basin region Malaria is a complex disease with many genetic and environmental determinants influencing the observed variation in response to infection, progression and severity. Several factors important for these different phenotypes include the parasite genetic make-up and host age, state of immunity and genetic background Plasmodium infection (see NOS2A) genes , southeast asian ovalocytosis and the Duffy-negative blood group are examples of the general principle that different populations have selected different genetic variants to protect against tion see , and ABOtion see , Toll-lition see , and theP. vivax malaria has been observed in five endemic states of the Brazilian Amazon P. falciparum infection in individuals from an endemic area in the state of Amazonas P. falciparum infections P. vivax infected patients may be unaltered by polymorphism in the promoter region of IL-10 gene Previous genetic studies in Brazilian Amazonian populations have demonstrated different malaria protective effects from blood-related polymorphisms  malaria in a population of the Brazilian Amazon. We genotyped 64 single nucleotide polymorphisms (SNPs) in 37 human host genes, including loci related with erythrocytes receptors and immune response. Our study is the first to comprehensively survey important malaria candidate polymorphisms (including HbS and ABO) in a Brazilian population from the Medium Negro River Basin in the Amazon. This setting provided an excellent field to test hypotheses on the generalization of established malaria-genetic associations, particularly for low endemic areas. It also provided a mean to find other associations related to either different mechanisms or by linkage disequilibrium with putative markers associated with susceptibility/resistance to Plasmodium infection Here, we investigated the association of a larger number of host candidate genes polymorphisms with susceptibility/resistance to Approval for the recruitment of participants, collection of blood samples, DNA preparation and DNA genotyping was provided by the relevant research ethics committee  and an informed consent was obtained from each participant.Potential participants were engaged between January 2002 and October 2006 from an ongoing epidemiological study of malaria in the cities of Barcelos (n = 596) and Santa Isabel do Rio Negro (n = 52), which are 350 km apart. The municipalities are located within the Negro river micro region in the state of Amazonas and display similar demographics. In 2006, the Annual Parasite Index (API) was 264.4 cases per 1000 inhabitants in Barcelos The individuals included in this study had lived in the study area for at least five year with similar social and genetic backgrounds. The population of both municipalities is predominantly of Amerindian descent from Tukano-Oriental speaking societies Plasmodium-infected cases were defined as an individual without symptoms for malaria within 30 days before or after the blood collection, but with a positive thick smear and/or PCR, following the recommendations of the Brazilian consensus group for studies of asymptomatic individuals.Three definitions of malaria cases were used for the association analysis: clinical malaria , asymptomatic infected individuals (n = 122) and individuals with a previous history of mild malaria (non-asymptomatic infection), but not infected at the moment of blood collection (n = 300). Clinical malaria was defined in accordance to the guidelines of the World Health Organization (WHO) for the American region and the Brazilian National Malaria Control Programme (PNCM) including symptoms associated to malaria  and a positive thick smear or rapid diagnostic test (RDT). Asymptomatic Plasmodium species was identified by PCR according to published protocols P. falciparum and P.vivax  The sample collection consisted of 535 cases  and 113 healthy controls with no previous history of malaria . DNA samples were prepared by extraction from 300 µl of total blood using a commercial kit following the manufacturer’s protocol (Promega®). Genomic DNA samples underwent whole genome amplification by Primer Extension Pre-amplification (PEP) before genotyping on a Sequenom® MassArray genotyping platform  with the never_malaria group (control); b) clinical_malaria group  with the never_malaria group; c) asymptomatic infection group with the never_malaria group and d) clinical_malaria group  with asymptomatic infection group.Association studies were performed comparing different groups: a) P-values from these correlated tests are reported. Haplotypes were estimated using an expectation-maximization algorithm 2 and D-prime metrics.Genotypic deviations from the Hardy-Weinberg equilibrium (HWE) were assessed using a chi-square statistical test. SNPs were excluded from the analysis if there was at least 10% of the genotype calls missing or a significant deviation from the HWE (p<0.0001). A case-control association analysis using SNP alleles and genotypes was undertaken by logistic regression and included site (Barcelos or Santa Isabel do Rio Negro) and gender as covariates. In this approach, the SNP of interest was modeled assuming several related genotypic mechanisms  and the minimum Fst metric Population structure or differences in allele frequencies between sub-populations can lead to false positive associations. Differences in allele frequencies were estimated between Barcelos and Santa Isabel do Rio Negro using an http://www.r-project.org). Performing multiple statistical tests lead to an inflation in the occurrence of false positives and, by using a permutation approach that accounted for correlation between markers and tests, the estimated p-value cut-off of 0.003 was considered statistically significant.Statistical analysis of SNPs on the X chromosome was performed for each gender separately and, where applicable, the results were pooled using meta-analytic techniques. All analyses were performed using the R statistical package  or due to a high rate of missing genotype calls . any_malaria versus never_malaria group: rs1800896– IL10-1082 , rs2706384 - IRF1 , rs2242665 - CTL4 , rs4986790 - TLR4  and rs909253 - LTA+252  , rs2242665 - CTL4 , rs4986790 - TLR4  and rs909253 - LTA+252 . When analyzing clinical_malaria versus asymptomatic infection group, associations were observed with the TNF-376 promoter SNP , previously associated in a number of other malaria studies. , rs1800896 - IL10-1082 , rs3024500  and rs1800890 - IL10-3533 . There was a high linkage disequilbrium (LD) between the three IL10 polymorphisms (minimum pairwise D’  = 0.85). A haplotype analysis of these three polymorphisms  revealed that those with the GCT allelic combination (∼10% frequency in population) were at a lower risk of any form of malaria  when compared to the common ATA combination  see . Four of−, which is a deficiency surrogate that occurs at a low frequency in our population (<3%). Two other candidate polymorphisms with a strong presence in the malaria literature, type HbS and B blood groups, were present at low frequencies in our population (<4%) and, therefore, lacked the power to detect an association  located in the CTL4 gene, displayed a significant association with reduced risk for clinical (mild) malaria. This gene encodes for a possible sodium-dependent transmembrane transport protein involved in the uptake of choline by cholinergic neurons. As the MHC class III region has a complex haplotype structure with long-range LD patterns, this finding could arise from a functional variant in high linkage with this SNP.Plasmodium infections.Polymorphic variability in the innate immune response gene IL-10 also showed a strong haplotype risk association  and beta (LT-ß/LTB) genes, which are closely related. The TNF and LTA genes are implicated in the host defense and pathogeneses of severe malaria In addition, the TNF-376 promoter SNP (rs1800750) was identified to have an association with clinical malaria . An assoP. falciparumP. falciparum infection both in healthy adults and children displaying severe and uncomplicated malaria P. falciparum infection levels Other cytokine-related findings include one SNP (rs2706384) in the Interferon Regulatory Factor 1 gene (IRF1), which was associated with an increased risk for clinical malaria. IRF1 is a critical mediator of IFN-γ activity and is crucial in both innate and adaptative immune responses against Finally, a polymorphism was seen in the TLR4 gene (rs4986790) that was associated with reduced risk for clinical malaria. Previous studies in an Amazonian population reported no association between TLR4 and mild malaria Plasmodium infection and malaria outcomes. To understand which are the real causal variants, re-sequencing of LTA, TNF and CTL4 genes and the surrounding MHC class III in a range of populations will be necessary to assist the design of large scale epidemiological studies. Previous candidate polymorphisms have arisen mostly from studies in an African setting, where the linkage disequilibrium or correlation structure between SNPs, the absence of Plasmodium vivax, endemicity, and disease severity complicate their relevancy to Amazonian populations. For example, the data analysis from our study revealed no significant associations for gene polymorphisms of the sickle-cell trait, blood-related polymorphisms and G6PD A- surrogate, which display inherited innate resistance to malaria. These results were not unexpected, given that the low frequencies of these alleles in the Brazilian population together with the current sample size that restricted the resolving power of the analysis to detect associations.Overall, this study represents the first association study from an Amazonian population involving a large number of host genetic polymorphisms with susceptibility or resistance to The issue of malaria disease association mapping and its implications for disease management in multiple geographic locations remains a major challenge confronting the field. The differences highlighted here and the association of specific SNP polymorphisms with clinical malaria in the Amazon basin support the need for additional studies. The results suggest a need for associations studies with more dense mapping of candidate genes, especially CTL4 and IL-10, along with other genes from the major histocompatibility complex region to identify additional functional variants most relevant for malaria in Amazonian populations.Table S1List of all polymorphisms genotyped in this study.(XLS)Click here for additional data file.Table S2X chromosome analysis.(XLS)Click here for additional data file.Table S3Allele frequencies differences between Santa Isabel do Rio Negro and Barcelos.(XLS)Click here for additional data file.Table S4Allele frequencies and test of association - Any_malaria versus never_malaria group.(XLS)Click here for additional data file.Table S5Allele frequencies and test of association - Asymptomatic versus never_malaria group.(XLS)Click here for additional data file.Table S6Allele frequencies and test of association - Clinical_malaria versus never_malaria group.(XLS)Click here for additional data file.Table S7Allele frequencies and test of association - Clinical_malaria versus asymptomatic group.(XLS)Click here for additional data file.


Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF). The in vivo targets of DENV and the relation between the viral burden in these cells and disease severity are not known.The levels of positive and negative strand viral RNA in peripheral blood monocytes, T/NK cells, and B cells and in plasma of DF and DHF cases were measured by quantitative RT-PCR.Positive strand viral RNA was detected in monocytes, T/NK cells and B cells with the highest amounts found in B cells. Viral RNA levels in CD14+ cells and plasma were significantly higher in DHF compared to DF, and in cases with a secondary infection compared to those undergoing a primary infection. The distribution of viral RNA among cell subpopulations was similar in DF and DHF cases. Small amounts of negative strand RNA were found in a few cases only. The severity of plasma leakage correlated with viral RNA levels in plasma and in CD14+ cells.B cells were the principal cells containing DENV RNA in peripheral blood, but overall there was little active DENV RNA replication detectable in peripheral blood mononuclear cells (PBMC). Secondary infection and DHF were associated with higher viral burden in PBMC populations, especially CD14+ monocytes, suggesting that viral infection of these cells may be involved in disease pathogenesis. Infection with dengue viruses (DENV) causes a wide range of manifestations from asymptomatic infection to a febrile illness called dengue fever (DF), to dengue hemorrhagic fever (DHF), a viral hemorrhagic disease characterized by plasma leakage and bleeding diathesis. The in vivo cellular targets of DENV remain to be completely identified. Studies of human dengue cases have identified macrophages and lymphoid cells as major targets of DENV as supported by the presence of viral antigen and RNA. Written informed consent was obtained from the parent or the legal guardian of each study subject. The study was approved by the Institutional Review Boards of the Thai Ministry of Public Health, the US Army Surgeon General's Office and the University of Massachusetts Medical School.The study design has been previously described. Samples were selected from archived specimens of clinically and serologically characterized cases. Samples were selected to include a representative subset of subjects from the parent study with sufficient specimens available for isolation of PBMC subsets. To provide data on changes over time in DENV RNA levels, we limited our selection to cases with samples available from multiple (at least 2) days during the febrile period. Samples from DF and DHF cases, cases with primary and secondary DENV infection, and viral serotypes were randomly selected. Twenty cases of DF and fifteen cases of DHF were selected . The cli6cells/ml. Cells were gradually cooled at −70°C for 18 hours and then stored in the vapor phase of liquid nitrogen for long term storage. One aliquot containing 1–2×106 cells was preserved in RNA preserving solution and stored at −70°C. PBMC were isolated from whole blood by Ficoll-Hypaque density gradient procedure. Cells were washed twice with RPMI and resuspended in freezing media containing 20% fetal calf serum, 10% DMSO in RPMI at 1–2×10Fractionated cells were lysed in Trizol solution  and RNA was extracted according the manufacturer's instructions. RNA was also extracted from 100 µL of plasma. Reverse transcription and PCR for positive strand DENV RNA were performed as described. 5′TGCTCGCGCTACTCTCTCT3′) and B2M-reverse (5′TCCATTCTCTGCTGGATGAC3′) in RT-PCR containing Sybr green . The cell numbers were estimated based on a standard curve generated with known numbers of PBMC. Cell-associated DENV RNA levels were expressed as copies/million cells following the formula: copies/million cells = ×PCR volume (ml))/cells per PCR)×106. All samples were assayed in duplicate and the mean values were used for analyses. The ABI gene detection system 7700  was used for PCR cycling amplification, data collection, and analysis.The number of cells in each PCR reaction was estimated by quantitative RT-PCR for β2-microglobulin mRNA using primers- B2M-forward . As shown in 2 = 0.474, P = .001), multivariate analysis showed that these parameters independently correlated with pleural effusion index (P = .001). Nadir platelet counts showed an inverse correlation with viral RNA levels in CD2+ cells. Viral RNA levels in plasma or cell fractions did not correlate with hemoconcentration or hepatic transaminase levels.We examined the correlations between viral RNA levels and parameters of disease severity. The levels of viral RNA in plasma and CD14+ cells on fever day-2, but not in CD2+ or CD20+ cells, correlated with the size of pleural effusions, an indicator of the severity of plasma leakage . In agreOur results demonstrate that peripheral blood B cells, T cells, and monocytes harbor DENV genomes during acute infection. The DENV RNA content associated with each of these cell populations was low (less than 1 copy/cell), however, indicating that no more than a small minority of cells within each fraction contains DENV in vivo. Consistent with this finding, we have been unable to detect DENV infected peripheral blood cells by flow cytometry (unpublished observation). Our results are in agreement with a previous study demonstrating low viral detection and recovery rate from PBMC. We found that B cells harbored the highest viral RNA levels among the cell types studied. This is consistent with other studies identifying B cells as the major cell population in peripheral blood that expressed DENV antigens and harbored infectious virus. High plasma viral load has been associated with increased severity of dengue illness. Studies have demonstrated that monocytes, B cells, and activated T cells can support DENV replication in vitro. We observed a decline in plasma DENV RNA levels at the time of defervescence as previously reported. 2 = 0.474) between viral RNA levels in plasma and in CD14+ cells. Therefore, the relative contributions of virus in these two compartments to plasma leakage could not be clearly delineated. It is also possible that the viral RNA levels in these two compartments are mechanistically related and both may contribute to plasma leakage. Similar correlations were not observed between viral RNA levels and hemoconcentration. However, hemoconcentration can be obscured by hemorrhage and fluid treatment, both of which are more common in DHF cases. We also observed an association between nadir platelet counts and CD2+ cell viral load. CD2+ T cells including NK and T cells may be activated by virus-antibody complexes and mediate antibody-dependent cytolysis directed against platelet or platelet progenitors expressing viral antigens on their surface.Our study demonstrated several correlations between indicators of disease severity and viral RNA levels in plasma or PBMC populations. Interestingly, indicators of plasma leakage  showed statistically significant associations only with viral RNA levels in plasma and CD14+ cells. The selective correlation of plasma leakage with viral RNA levels in CD14+ cells suggests that these cells may play an important and direct role in this disease process. DENV-infected monocytes/macrophages have been shown to produce proinflammatory cytokines with permeability-enhancing effects. Our findings are subject to several limitations. The number of cells required for cell sorting and quantitative RT-PCR analysis limited the number of cases that could be studied. However, the clinical and demographic characteristics of the selected cases did not significantly differ from those of the total dengue cases enrolled during the same period, suggesting that we did not introduce any bias by our selection. The paucity of DHF cases with a primary infection precluded the analysis of a relation between clinical severity and viral RNA levels in primary infections. The small number of samples representing each viral serotype in this study might have limited our ability to detect serotype-associated differences in DENV RNA distribution among cell populations. In addition, our assay method would not differentiate between surface and intracellular virus, a distinction that may have implications on cell signaling and cytokine responses. Studies have shown that the interaction of virus-antibody complexes with Fc receptors may induce the production of inhibitory cytokines, such as IL-10. Table S1Patient characteristics of the all dengue cases enrolled between 1994–2001 and dengue cases selected for this study.(DOCX)Click here for additional data file.Table S2Patient characteristics of dengue fever (DF) cases with either primary or secondary infection.(DOCX)Click here for additional data file.Table S3Primers used in positive and negative strand RT-PCR.(DOCX)Click here for additional data file.Table S4DENV RNA in C6/36 cells detected by positive and negative strand PCR.(DOCX)Click here for additional data file.


Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC.Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression.KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines.Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy. Pancreatic ductal adenocarcinoma (PDAC) is an insidious disease, with the shortest survival of any solid malignancy in vitro cell culture models to whole animal in vivo models. In vitro models offer advantages such as efficient derivation of data, control over drug delivery, lower cost and reproducibility of results, allowing for high-throughput analysis of multiple cell lines. However, two-dimensional culture poorly recapitulates in vivo biologic behavior and drug delivery, and undermines the impact of the tumor microenvironment. Thus, we have demonstrated in our laboratory that in vitro assays do not correlate with response in the orthotopic xenograft model for targeted therapies to focal adhesion kinase (FAK) Accordingly, various preclinical models have been established to study PDAC, ranging from simple in vivo models have been developed KRAS plus deletions or mutations in P53P16INK4MISTSMAD4TGFβDue to these limitations, several Invasive approaches have been used in murine models of PDAC, where cells or tumor pieces are implanted heterotopically (subcutaneous) in vitro cell culture, they often are not representative of their original human source tumor While each of these models has potential value, we believe that orthotopic xenograft models are the best for studying novel therapeutic approaches to PDAC because such models more closely recapitulate human disease Collection of human PDAC specimens was performed with approval of the Institutional Review Board at the University of Virginia in coordination with the Biorepository and Tissue Research Facility. All patients provided written consent for participation and no patients received neoadjuvant therapy. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health 1 and F2 generations. For propagation of the tumor line beyond these first two generations, athymic nude mice were used, as they retain innate immunity , which is impaired in NOD SCID mice. Mice were housed in pathogen-free conditions, acclimated to their new surroundings for at least 48 hours prior to tumor engraftment, and maintained in accordance with institutional standards. All animal surgery was performed under 2,2,2-tribromoethanol anesthesia (4 mg/10 gm body weight). Post-surgery mice were administered ketoprofen 0.1 mg for pain control and were observed continuously for signs of pain or distress  until they recovered from anesthesia, then monitored daily for 48 hr for signs of pain or distress. Humane endpoints were observed throughout experiments with mice being sacrificed when tumors reached a volume greater than 1500 mm3 by MRI assessment or when mice developed 15% weight loss. Mice were sacrificed via isofluorane anesthesia followed by cervical dislocation.Following resection and pathological review of the patient tumor, residual tumor tissues were collected and placed in Roswell Park Memorial Institute media (RPMI) for surgical transplantation (below) or cryopreservation in fetal bovine serum (FBS) with 10% DMSO . Six to 0) or earlier generation xenograft . A 1.5-cm left flank incision was used to access the peritoneum of anesthetized mice, the pancreas was exteriorized using a sterile cotton swab and a small piece (∼25 mm3) of fresh patient tumor (F0 tumor) was sutured onto the pancreas using 5-0 Prolene . The pancreas was repositioned and the wound closed using 4-0 Vicryl suture (Ethicon).Human tumors were surgically implanted onto the pancreata of mice immediately following resection from either a patient (Fin vivo by volumetric magnetic resonance imaging (MRI). For MRI, mice were anesthetized and 0.5 mm axial imaging slices were generated, encompassing the entire tumor. Tumor area was measured for each individual image slice and tumor volume was calculated using the following: VolumeTUMOR = (AreaIMAGE1+AreaIMAGE2+AreaIMAGE3…) as described previously Tumor size was determined 3 as measured by MRI at which point the mice underwent necropsy with complete tumor extirpation. Fresh tumor samples (approximately 50–60 mm3) were either: preserved in Allprotect  and stored at −80°C, snap-frozen in liquid nitrogen and stored at −80°C, cryopreserved in FBS with 10% DMSO; or fixed in formalin for subsequent analysis. Small pieces (∼25 mm3) were placed freshly in RPMI for reimplantation into the next generation of mice.Xenograft tumors were allowed to grow to a size of approximately 400–500 mm0) and mouse  tumors. A board-certified pathologist specializing in pancreatic and liver pathology (EBS) reviewed all slides, assessing tumor architecture and desmoplastic content and made qualitative comparisons between human and mouse tumors. Tumors were defined as having “abundant” desmoplasia when stroma comprised more than 50% of the cross-sectional tumor area.Tumor pieces were placed in tumor blocks, fixed in zinc-buffered formalin for 24 hours, embedded in paraffin, and stored at −80 C. Hematoxylin/eosin staining was performed on human , and from frozen tissue samples preserved in Allprotect tissue reagent using the Allprep kit (Qiagen). DNA was quantified using a spectrophotometer and diluted to 50 ng/µl for PCR amplification, using Bio-X-Act™ short mix  in a Techgene  or Mastercycler gradient  machine. The presence of mutation was assessed for the following genes: 0), normal human pancreas and four established and previously described For gene expression profiling, tumor xenografts were analyzed and compared to original patient tumors , including human phospho-receptor tyrosine kinase (RTK) and cytokine arrays, measured the activity of various signaling pathways. Lysates were prepared using NP-40 lysis buffer with leupeptin, aprotinin, sodium orthovanadate, and EDTA. Total protein concentration was determined using the Pierce BCA Protein Assay , using 250 µg of lysate for each array. Arrays were developed using Pierce ECL Substrate  and analyzed using a Bio-Rad GS-800 densitometer  and ImageQuant TL 2005  software.Phospho-RTK and cytokine expression were normalized to the maximum value of the negative control for each array as follows. The final value was calculated by determining the average density of each sample and subtracting the standard error of the mean and the maximum value of the negative controls (i.e. background). Data are reported as multiples of background signal for each receptor or cytokine.p-values are two-tailed, and significance indicated by p-values<0.05. Association between time to establishment of F1 tumors in mice and patient survival time was analyzed with a non-parametric Spearman rank-order correlation analysis. GraphPad Prism software  and R statistical software  were used for all statistical analyses.For statistical analyses, group comparisons were unpaired. Categorical variables were compared using Fischer's exact, McNemar's, or Pearson's Chi square tests, as appropriate. Analysis of variance compared continuous variables. Categorical variables are expressed as percentages of the group of origin, and continuous variables as means ± standard deviation. Survival curves were generated using the Kaplan-Meier method and analyzed using the log-rank test. All 1) have successfully grown in mice; of which greater than 95%, were also successfully propagated in subsequent generations  of mice (data not shown). Patient tumors (F0) derived from metastatic patient lesions were more likely to grow  compared to patient-derived primary pancreatic tumors ; the former were established more quickly , and were associated with increased incidence of liver and peritoneal metastasis in F1 mice .To date, 47% (21/45) of initially implanted tumors  and, strikingly, more highly correlated for successfully propagated tumors . Patients whose F1 tumors grew to 400–500 mm3 in less than four months after implantation had a median survival of 6.3 months, compared to 13.7 months when F1 tumors took 4 months or longer to grow, and 20.6 months when tumors failed to grow .Time to establishment of Fsurvival . Patientp = 0.48) nor stromal content (McNemar's p = 0.25) differed significantly between mouse xenograft tumors and their respective F0 patient tumors , while two tumors had low stromal content in F0 generation, but exhibited a higher level of stroma in F1. Both differentiation state and stromal content was preserved among early as well as late passage mouse tumors  with all mutations occurring in codon 12 and none in codons 13 or 61. p53 mutations were present in 11/15 tumors (73%), involving various codons within exons 5, 6, and 8 SMAD4 mutations were present in 7/15 tumors (47%), involving sequence alterations in exons 8, 9, 10, or 11. No BRAF mutations were observed. BRCA2 missense mutations were observed in 2 of 15 tumors (13%).PCR amplification and DNA sequencing of DNA from 15 individual F1 tumors were assessed for activation (phosphorylation) of 47 RTKs (p = 0.0423) and higher concentrations of cytokines  compared to poorly differentiated tumors, again emphasizing the importance of studying genetically diverse human tumors and pointing to the influence of differential gene expression on the tumor cell-microenvironment interactions.Whole tumor lysates from Fp = 0.0256).Gene expression profiling was performed for xenografted tumors, established cell lines, and normal human pancreatic specimens. Unsupervised clustering is shown as a dendrogram in 0) with xenografted tumors (F1 to F7) demonstrated significantly higher correlations in gene expression across ∼>40,000 probe sets with the average (Pearson) correlation coefficient 96% (93∼98%) than comparison of two independent xenograft models with the average correlation 93% (89∼95%)  and greater than 95% grow in subsequent generations. Interestingly, the time to initial tumor engraftment to a size of 400–500 mm3 in the mouse pancreas correlated with patient survival. In addition, patient-derived metastatic tumors were more likely to grow in F1 mice. This suggests that more aggressive patient tumors also grew more aggressively as mouse xenografts in this model. This parallels studies which demonstrated that patients whose non-small cell lung cancers successfully engrafted into mice had significantly shorter disease free survival, compared to patients whose tumors did not establish Efficient establishment and propagation of tumors is essential in any xenograft model. In the model described here, nearly half of original F0) were compared to F1 tumors and subsequent tumors passaged through multiple generations. However, one limitation of our approach is the lack of comparison of the tumors to a larger dataset containing unmatched patient tumors and mouse xenografts. Despite this limitation, to our knowledge, this is the first preclinical pancreatic cancer model to display such highly conserved gene expression. The diversity of oncogenic drivers of tumor progression is one of the greatest strengths of the xenograft model and one of the key limitations of engineered cancer models. Patient derived xenografts exhibit multiple, well studied genetic mutations common to human PDACs — KRAS, P53, and SMAD4To be adequate representations of human cancers, xenograft models must accurately reflect the histopathologic and molecular features as well as the diversity of human tumors. In the model described above, orthotopically propagated mouse tumors mimic the architecture and stromal content of their respective human tumors, maintaining tumor grade through multiple passages, similar to previous observations in lung and breast cancer models Because metastatic lesions provide the greatest therapeutic challenge and impact on survival, an ideal tumor model should recapitulate human metastatic patterns. While subcutaneous injection and spontaneous tumor models are limited or variable in this regard in vitro and assessed its effects on tumor growth and metastasis in an orthotopic murine model in vitro cell culture assays. Treatment of orthotopic tumor bearing mice with PF-562,271 leads to a significant inhibition of tumor growth and reduces the number of tumor associated stromal cells, indicating that inhibition of adhesion signaling reduces pancreatic cancer cell growth either directly by contributing to inhibition of cell proliferation and/or by altering the cellular composition of the tumor microenvironment. Thus, the use of preclinical tumor models with a relevant tumor microenvironment will be essential in making progress in cancer therapeutics.The impact of the tumor microenvironment cannot be overemphasized, as it influences drug delivery, is an important source of tumor growth factors and contributes to tumor survival in the face of therapeutic treatments. The cytokine array data (While no perfect model exists for PDAC and important data may be garnered from each model, the orthotopic xenograft model using a diverse array of freshly implanted human tumors provides a unique platform for assessing tumor responses to therapeutic strategies. The orthotopic xenograft model using fresh human specimens most closely recapitulates the molecular and genetic heterogeneity of PDAC, human metastatic patterns, the tumor microenvironment, and drug delivery. This model holds great promise for identification and testing of additional rational therapeutic approaches for pancreatic cancer and the selection of personalized cancer therapies, and provides the opportunity for the identification of genetic and molecular biomarkers of response to therapy.