Impacts of human land use pose an increasing threat to global biodiversity. Resource managers must respond rapidly to this threat by assessing existing natural areas and prioritizing conservation actions across multiple spatial scales. Plant species richness is a useful measure of biodiversity but typically can only be evaluated on small portions of a given landscape. Modeling relationships between spatial heterogeneity and species richness may allow conservation planners to make predictions of species richness patterns within unsampled areas. We utilized a combination of field data, remotely sensed data, and landscape pattern metrics to develop models of native and exotic plant species richness at two spatial extents (60- and 120-m windows) and at four ecological levels for northwestern Ohio’s Oak Openings region. Multiple regression models explained 37–77 % of the variation in plant species richness. These models consistently explained more variation in exotic richness than in native richness. Exotic richness was better explained at the 120-m extent while native richness was better explained at the 60-m extent. Land cover composition of the surrounding landscape was an important component of all models. We found that percentage of human-modified land cover (negatively correlated with native richness and positively correlated with exotic richness) was a particularly useful predictor of plant species richness and that human-caused disturbances exert a strong influence on species richness patterns within a mixed-disturbance oak savanna landscape. Our results emphasize the importance of using a multi-scale approach to examine the complex relationships between spatial heterogeneity and plant species richness. Biodiversity is increasingly threatened by growing human impacts throughout the biosphere  is frequently used to measure biodiversity  occurs near the eastern extent of the historic Midwest Oak Savanna region  modified-Whittaker, multi-scale plot  to the nearest 1 %, along with bare ground, litter (attached), duff (detached), coarse woody debris, cryptobiotics , and tree/shrub canopy (>1.7 m). Cover for species occupying <1 % of a 1-m2 subplot was recorded as 0.5 %. Due to layering of foliage, litter, duff, and cryptobiotics, it was possible for cumulative cover to exceed 100 %. We recorded cumulative number of plant species within each of the 10-m2 subplots, the 100-m2 subplot, and the 1,000-m2 plot. Within each 1,000-m2 plot, we recorded by species all woody stems ≥2.5 cm dbh (diameter at breast height). All upland communities were sampled from 26 July to 20 September 2008 and from 2 August to 22 September 2009, corresponding to peak biomass for these communities. For wet prairies, sampling occurred from 23 May to 2 July 2009, coinciding with availability of flowers and fruits within the Family Cyperaceae (necessary for their successful identification) rather than onset of peak biomass within these sedge-dominated communities. Therefore estimates of cover could not be directly compared between upland and wet prairie communities. For all communities, species were classified as either native or exotic to our study area following Andreas and others  to a depth of 40 cm using a 2.5-cm diameter soil probe after removing any surface litter. For each plot, soil samples were pooled into a single sample following Kumar and others  and air 2 plot to nearest patch edge, paved roadway, water source (dug pond or drainage ditch), and human dwelling using high-resolution color orthophotos of our study area  and measured the following variables using ArcGIS 9.1 Spatial Analyst : mean elevation (m), slope (%), and aspect (radians) transformed into north–south and east–west gradients Patch Richness Density: measures number of different patch types present per total landscape areaShannon’s Diversity Index: measures the proportional abundance of each patch type on the landscapePercentage of Landscape: measures total area of all patches of the corresponding patch type per total landscape areaWe evaluated landscape heterogeneity at each study site by measuring selected landscape pattern metrics at two nested spatial extents (60-m and 120-m) surrounding each 1,000-mP = 0.05. For all variables, we tested for normality within the residuals using the Shapiro–Wilks test and examined residual plots for obvious patterns indicative of heteroscedasticity. Data were transformed when appropriate prior to analysis to reduce the influence of non-normality/heteroscedasticity within the datasets  transformation for count data). Data exhibiting strong non-linear relationships following transformation were excluded from linear regression analyses.Our general statistical approach was to test for linear relationships between native or exotic plant species richness (response variables) and selected physical/landscape variables  at each level within the Oak Openings region ecological classification hierarchy Fig.  and thenx and y variables, giving a corrected F value and corresponding P value for each linear regression model  to develop a set of candidate models of native/exotic richness at both 60-m and 120-m spatial extents within each of the four levels of the Oak Openings ecological hierarchy. Before conducting multiple regression analyses, we examined all predictor variables for cross-correlations and multicollinearity by evaluating correlation matrices and inverse correlation matrices of each set of predictor variables. Any variables with cross correlations >±0.75 or those with variance inflation factors >2.5 were not included in the same model  to select the “best” model among all possible candidate models for native and exotic richness at both 60-m and 120-m spatial extents. Only candidate models with Δ AICc of ≤2 were given consideration . Thirty out of 39 research plots occurred within existing managed preserves including all oak savanna, mesic prairie and wet prairie plots; while four of eight dry prairie plots and two of seven sand barrens plots occurred within managed preserves. While the effects of specific management and restoration treatments could not be evaluated from our data, the fact that oak savanna, mesic prairie, and wet prairie sample sites were not found outside of existing managed preserves within our sampling design supports existing evidence that large-scale intact remnants of these communities do not persist in the Oak Openings region without regular ecological management treatments such as prescribed fire , including 48 species listed as endangered, threatened, or potentially threatened in Ohio ODNR . This acutilleul ). Among P < 0.05; Table At the region level , there were no significant relationships between native richness and the physical/landscape variables that we measured , a likely source of soil sodium through runoff of road salt.Within the heavily fragmented Oak Openings region, we found ample evidence of the influence of human-caused disturbances associated with habitat fragmentation. Among landscape variables, we found that exotic species richness was positively correlated with amount of patch edge (measured by Landscape Shape Index), relative number of patches on the landscape (measured by Patch Richness Density and Shannon Diversity Index), proximity to roads, and proximity to man-made ditches/ponds; but was negatively correlated with patch connectedness (measured by Cohesion Index). Among soil nutrients, we observed a positive correlation between native richness and soil organic carbon among upland sites, which may be related to the well-established effects of soil disturbance on reducing soil organic carbon  which were not evaluated in our study.Previous studies have established a clear justification for using plant species richness as a basis for measuring ecosystem restoration success, both theoretically Wang  and in p


Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control. Plasmodium parasites transmitted to their human hosts by the bite of anopheline mosquitoes. Malaria has been charged with causing more human deaths than any other disease in human history and continues to kill about 660,000 annually [Plasmodium to antimalarial drugs. Sequencing the genome of the main malaria vector, Anopheles gambiae [Plasmodium. Altering the mosquito genome in a way that abates Plasmodium transmission, through transgenesis or other sophisticated genetic engineering tools, can offer new perspectives in the fight against malaria. On one hand, experimentally altering mosquito genes of interest will advance our fundamental understanding of the biological interactions between mosquito and parasite, and may help target vulnerable points in the parasite cycle. On the other hand, the prospect of releasing engineered male mosquitoes to propagate malaria resistance genes through wild susceptible vector populations has been receiving increasing attention [Malaria is caused by annually . A reduc gambiae , enabledttention ,4. NovelXanthomonas Transcription Activator Like Effector (TALE) proteins [Aedes mosquitoes using this method [TEP1 gene, a key component of the mosquito immune system.Recently, a novel class of DNA-binding protein domain derived from the proteins ,6 has beproteins ,8. Theseproteins ,10), higproteins  making tproteins . Mutatioproteins –19. Verys method . In rices method . We hypoAnopheles gambiae , we expected that mutant recovery after direct injection of TALEN-encoding plasmids or mRNA might be difficult. For this reason and to enable controlled mutagenesis experiments, we preferred transgenic expression of the TALENs in the mosquito germ cells. To obtain the proof-of-principle for gene targeting in A. gambiae via transgenic TALENs, we selected the well-characterized immune gene TEP1 as a target. TEP1, a protein similar to vertebrate complement factor C3, binds Plasmodium parasites as they invade the mosquito intestine and kills them in a manner probably dependent on its thioester site located in the C-terminus [TEP1 mutants will be instrumental in further dissecting the antiparasitic complement-like system in mosquitoes.Since the embryo microinjection procedure is technically challenging in terminus –25. TEP1FokI domains of the TALENs to properly dimerize and create a double-stranded break. Cleavage of the bound DNA molecule occurs near the center of the sequence separating the two target sites. We designed a single pair of TALENs to target a site within the TEP1 gene centered on an NcoI restriction site (5’-CCATGG-3’), offering the possibility to easily screen individual mosquitoes for mutations that destroyed the NcoI site . This figure is a conservative estimate of mutation frequency, as we subsequently observed some mutations that left the NcoI recognition sequence intact. In order to obtain a mosquito population containing a higher frequency of mutations, we self-crossed successive generations of mosquitoes expressing both TALENs of the pair. This was facilitated by automated COPAS selection of larvae [TEP1. This indicated that the frequency of mutations increased faster than predicted if mutations accumulated linearly from one generation to the next. This observation is consistent with a model where mosquitoes already carrying a heterozygous mutation employ homologous recombination to repair new TALEN-induced breaks in the wild-type chromosome, thereby effectively copying the existing mutation onto the newly damaged chromosome. This suggests that in addition to NHEJ, TALEN-caused breaks can also be repaired by homologous recombination. Therefore, the observed number of NHEJ mutations is an underestimation of the true rate of TALEN activity.A distinct transgenic mosquito line was generated for each TALEN of the pair, the expression of which was driven by the germline . Therefoith NcoI . Of 310 f larvae  that hadf larvae . At leasTEP1. To investigate this, we sampled mosquito larvae from lines carrying a single TALEN maintained at high population levels for 8 (right TALEN) or 10 (left TALEN) generations, and again sampled larvae after about 16 generations. We expected that such a high number of generations would have allowed rare “monotalenic” mutational events to accumulate in the population. Out of 96 individual larvae tested by PCR for each sample, none carried a mutation in the TEP1 NcoI site. The observed absence of mutations among 576 haploid genomes exposed to single TALEN activity for 7, 9 or 15 generations suggests that single TALENs never or rarely induce mutations at their target site. To strengthen this point, we purified DNA from 2600 pooled larvae whose genomes had been exposed to one TALEN of the pair for 7 or 9 generations, and PCR-amplified the target region. PCR products appeared to be fully cleaved by NcoI, pointing to the absence of TALEN-induced mutations. To increase the chance of detection of a minor fraction of mutated products, we purified the region of the gel in which uncleaved PCR products may exist, cloned them into a plasmid, and examined E. coli transformants containing single copies of the PCR products. Again, all cloned fragments were cleaved by NcoI. Although we note that deep sequencing of amplicons would provide a more sensitive assay to detect rare mutations, this result further suggests that single TALENs rarely or never generate mutants. The apparent absence of single TALEN background activity was likely facilitated by our design scheme in which we used obligate heterodimeric FokI domains in TALEN construction [We wondered if a single TALEN of the pair is capable of causing mutations in truction ,31.TEP1 mutations were very similar in nature to mutations obtained in other organisms  and 2  amino-acids, respectively; line M∆3+6 lacks 3 endogenous amino-acids but gained an insertion of 6 exogenous ones, line M∆ct1 and M∆ct2 are frame-shift mutations causing the loss of the entire protein C-terminus  was achieved by injecting mRNA encoding Zinc Finger Nucleases [TEP1 as a first target; future work will make use of the obtained hypersusceptible mutant lines to further dissect the role of this important anti-malarial factor in parasite killing. Beyond the research field of mosquito immunity, this study paves the way for numerous other applications such as obtaining mutations in Anopheles genes considered to be essential for Plasmodium parasite development [Here, we obtained proof of principle that TALENs can be used for targeted mutagenesis in the genome of the malaria mosquito, which is notably difficult to manipulate genetically. Recently, Aryan et al.  reporteducleases . In bothelopment –39. Disrelopment . Of noteOur experimental protocols were approved by Comité de Qualification Institutionnel (CQI), the ethics evaluation committee of INSERM . Mosquitoes were reared and blood-fed on anesthetized mice in compliance with French and European laws on animal house procedures (agreement E67-482-2 of the Direction of Veterinary services of the French Ministry of Agriculture).ϕC31 attB site for genomic integration into transgenic lines harboring attP sites. The first module used in Golden Gate assembly provided the Vasa promoter characterized in [A. gambiae. The annotated sequence of the three plasmids is provided in TALENs targeting the sites shown in rized in . The lasrized in , designeϕC31 integrase-mediated transgenesis [A. gambiae lines X1 and X13, which are derived from laboratory strain G3. These two lines carry a PiggyBac transgene on chromosome II, containing an attP docking site. Individual transgenic larvae carrying the inserted left or right TALEN genes at the X1 or X13 attP site were identified by their red or yellow fluorescence, respectively. Resulting adults were crossed to their non-fluorescent parental line. In the F2 generation, fluorescent homozygous larvae were COPAS-selected [The TALEN-encoding vectors were inserted by sgenesis  into theselected  to estabTEP1 spanning the TALEN target site using Phusion or Phire Polymerases  and primer 5’-TCAACTTGGACATCAACAAGAAGGCCGA-3’ in combination with either 5’-GCATATCTTTGTGCCACACTTT-3’ or 5’-GCCACCGTAACGAATTTCCA-3’. The PCR product was digested with NcoI (Fermentas), the recognition site of which is centrally located in the sequence cut by the TALENs. PCR products corresponding to mutant TEP1 alleles were not digested by NcoI. These PCR products were sequenced directly in the case of homozygous individuals, or cloned  and subsequently sequenced.In putative mutants (mosquitoes arising from parents expressing both TALENs), we PCR amplified a region of TEP1 mutants, mosquitoes from this population were out-crossed to the parental line. The progeny carried a single TALEN and was therefore no longer subjected to TALEN mutagenesis. Among this progeny, we screened single adult mosquitoes by PCR on one leg using the Phire direct animal tissue PCR kit . The identified heterozygous mutants were individually crossed to the parental line. In the F2 progeny, we identified homozygous mutants by leg PCR and pooled them to start distinct homozygous mutant families.For mutagenesis experiments, the two TALEN lines were crossed. From the F2 generation onwards, 1000 mosquito larvae that inherited a single copy of each TALEN were COPAS-purified to initiate the next generation. To isolate TEP1 by double-stranded RNA injection into the thorax of adult mosquitoes and mosquito infections on mice carrying Plasmodium berghei GFP-con 259cl2 were performed as described [RNAi silencing of escribed . To semi2 [Whole-body mosquito extracts were obtained by grinding one adult female mosquito in 80 µl of protein sample buffer. The sample was denatured for 3 min at 95°C and centrifuged. 8 µl were loaded on 8% SDS-polyacrylamide gels. Hemolymph samples were prepared as described , 10 µl o2  or again2 .Text S1The nucleotide sequences of plasmids and building blocks used to construct the TALEN-expressing transgenesis vectors are provided. Building blocks not listed here are published in references (DOCX)Click here for additional data file.


Details on the 28-year treatment history of a patient with an endocrine-responsive breast cancer are provided. She was originally diagnosed as having a T1N0M0 cancer after a modified radical mastectomy at age 41. Fifteen years later, in 1998, she presented with hemoptysis and pleuritic chest pain: a 10 cm right atrial tumor and estrogen receptor (ER) positive endobronchial and adjacent lung parenchyma adenocarcinoma were documented. Epithelial markers normalized as she manifested a partial response (PR) lasting 3 years with tamoxifen treatment. From 2001 to 2007 she benefitted from exemestane treatment. Upon progression in the previous lung area and left adrenal, exemestane withdrawal led to transient decrease in markers. Six months later (in July 2008), with growth in her adrenal tumor, laparoscopic adrenalectomy was performed: in addition to ER positivity, the tumor showed Her2 overexpression and amplification. She has subsequently had some control of disease with fulvestrant, letrozole + trastuzumab, and subsequently letrozole + lapatinib. In addition to the chronicity of disease, this history illustrates the expanding range of treatments available for endocrine-responsive breast cancer commensurate to our greater understanding of tumor biology. Hormonal modulation in patients with estrogen receptor (ER) and progesterone receptor (PR) positive metastatic breast cancer is a treatment strategy resulting in less morbidity than chemotherapy. Sequential interventions in a woman initially diagnosed at age 41 with a T1N0M0 breast cancer in 1983 and recurring in 1998 are described. This case highlights how our understanding of endocrine-based therapy and breast cancer biology offer an array of treatment options for luminal A and B subtypes of metastatic breast cancer [This patient’s treatment is best described in five parts.Initial diagnosis to recurrence (1983–1998) This woman, without a family history of cancer but of Ashkenazi Jewish origin, had routine mammography leading to a diagnosis of an invasive ductal carcinoma (T1N0M0) of the right breast at age 41. After a modified radical mastectomy and axillary node dissection  she was given no adjuvant therapy. In 1998, she presented with complaints of right pleuritic chest pain, cough with hemoptysis and shortness of breath. Imaging and subsequent bronchoscopic biopsy revealed metastatic adenocarcinoma, ER and PR positive. A 10 cm lesion was noted in the right atrium. Her epithelial markers were elevated (Response to tamoxifen (1998–2001) and to exemestane (2001–2007) Complete regression of the right atrial lesion within 6 months ensued following tamoxifen treatment. Lung findings were stable until 2001 at which time a new right lung infiltrate was noted; treatment was switched to exemestane. She experienced improvement until February 2008 when routine labs showed increasing CEA and increased 18fluorodeoxyglucose (FDG) uptake on PET/CT was noted in the left adrenal  The patient’s CEA level dropped over 2 consecutive months  Laparoscopic removal of left adrenal tumor was achieved without incident in July 2008, as CEA dropped and remained within the normal range (<5) for 1 year. The adrenal tumor was a well-differentiated adenocarcinoma with strongly positive hormone receptors and Her2 3+ immunostaining; by FISH the Her2 had 4-fold amplification. In August 2009, as CEA began to rise, fulvestrant was started. Fulvestrant therapy was discontinued in May 2010 as CEA rose and imaging showed progression; a trial of anti-Her2 therapy combined with letrozole was instituted.Endocrine therapy (letrozole) combined with anti-Her2 therapy (2010–present) The patient eventually received letrozole and trastuzumab every 3 weeks. This treatment was discontinued on March 2011 when a surveillance PET/CT indicated a mixed response: increase in the right hilar mass, while the right upper lobe mass was smaller. Her markers have since continued to rise, accompanied by some weight loss and intermittent fevers. Additional anti-Her2 therapy including lapatinib and letrozole was started in June 2011. She and her husband continue to be reluctant to undergo trials with experimental agents; therefore, chemotherapy with capecitabine was started in August 2011 because of symptomatic progression in the lung and mediastinum.Most T1N0M0) invasive ductal carcinomas fall within the luminal A subtype N0M0 inva. The EarUpon recurrence, tamoxifen was impressively effective against our patient’s metastatic disease including normalization of the epithelial markers and complete regression of the right atrial tumor and this was followed by an even longer response on aromatase inhibitors (AI)—that had by then demonstrated superior outcome compared with tamoxifen in metastatic disease  as well The Breast concluded that location greatly influences prognosis. Liver metastases are commonly seen as part of overall progression; data regarding adrenal metastasis are lacking [The use of surgical resection in treatment of metastatic breast cancer rather than systemic treatment is increasingly being considered in patients with so-called ‘oligometastatases’. A 2004 review in  lacking .Our patient also benefitted from other endocrine interventions, such as the ER downregulator, fulvestrant. Mechanisms leading to endocrine resistance in hormone receptor positive breast cancer are under intense study –12. The


Peptide YY (PYY) and neuropeptide Y (NPY), two members of the pancreatic polypeptide-fold family of biologically active peptides, play important roles in the regulation of food intake, energy homeostasis and emotional-affective processes. While NPY also participates in nociceptive processing and cognition, the implication of PYY in pain, learning and memory has been little studied. Therefore, male wild-type, PYY knockout (PYY(−/−)), and NPY plus PYY double knockout (NPY(−/−);PYY(−/−)) mice were studied for their sensitivity to noxious heat in the plantar test and for their spatial learning and memory performance in the Barnes maze.In the plantar test mice were habituated in small compartments for 1 h. Subsequently the plantar side of the right and left hind paw was exposed to an infrared source through a glass plate (cut off time: 15 s). The withdrawal response was assessed at two infrared intensities, and the withdrawal latency recorded. In the Barnes maze test, mice were placed in the middle of a circular maze with 20 holes. The task of the mice was to find a target hole with an escape box within a preset time. The animals received 4 training runs on the first day and 3 trainings daily for the following 3 days. The first probe trial was performed on day 5 and the second probe trial on day 12, to check short-term and long-term memory, respectively. Target hole visits, preference  and latency (time taken to reach the target hole) were measured.In the plantar test at the higher infrared intensity, PYY(−/−) and NPY(−/−);PYY(−/−) mice presented with a significant decrease (p < 0.01 and p < 0.05) in withdrawal latency when compared with wild-type mice. At the lower infrared intensity, the withdrawal latency of PYY(−/−) mice was also significantly shorter (p < 0.01) than that measured in wild-type mice, whereas NPY(−/−);PYY(−/−) mice did not significantly differ from wild-type mice. In the Barnes maze test, significant differences were only found during probe trial 1 when NPY(−/−);PYY(−/−) mice visited the target hole less often (p < 0.01) than wild-type mice. In addition, the total number of hole visits made by NPY(−/−);PYY(−/−) mice was significantly (p < 0.01) lower than that made by wild-type animals.The most important observation of this study is that genetic deletion of the gut hormone PYY decreases the pain threshold to noxious heat. Additional knockout of the neuropeptide NPY, which is known to play a role in nociception, did not increase the hyperalgesic effect of PYY knockout. In contrast, the impact of PYY and NPY deletion on the performance in the Barnes maze was modest, and the decrease in target hole visits was probably due to reduced locomotion and did not reflect an impairment of short term memory.


Microcystis aeruginosa under different nutrient and light conditions. MALDI-TOF MS revealed alterations in the microcystins signatures under N and P poor conditions and high light intensities (150 and 400 μmol photons m−2s−1). Variations in the general oligopeptide composition were caused by a gradual disappearance of microcystins with low relative intensity signals from the fingerprint. The extent of such variations seems to be closely related to physiological stress caused by treatments. Under identical clonal compositions, alterations in the oligopeptide fingerprint may be misinterpreted as apparent shifts in chemotype succession. We discuss the nature of such variations, as well as the consequent implications in the use of cyanobacterial chemotyping in studies at the subpopulation level and propose new guidance for the definition of chemotypes as a consistent subpopulation marker.The occurrence of diverse oligopeptides in cyanobacteria, including the cyanotoxins microcystins, has been recently used to classify individual clones into sub-specific oligopeptide chemotypes, whose composition and dynamics modulate microcystin concentrations in cyanobacterial blooms. Cyanobacterial chemotyping allows the study of the ecology of chemotypical subpopulations, which have been shown to possess dissimilar ecological traits. However, the stability of chemotypes under changing abiotic conditions is usually assumed and has not been assessed in detail. We monitored oligopeptide patterns of three strains of  Freshwater cyanobacteria are well known for their occasional massive proliferation in temperate water bodies during late summer periods. Cyanobacterial blooms are of public health concern, due to the ability of many genera to produce a wide range of toxins and other bioactive compounds . An impoThe traditional cyanobacterial taxonomy following botanical criteria is based mainly on morphological features. However, the existence of transitional populations with wide morphological variations , togetheOligopeptides are produced by non-ribosomal peptide synthases (NRPS) , with thMicrocystis aeruginosa producing a wide range of microcystin variants and cultured under different scenarios. We focused on M. aeruginosa, a species that has been pointed out as the most important microcystin producer in Mediterranean latitudes  = 200 µM), while P-poor treatment (NP) consisted in a modified BG11 medium containing only 10% of the original K2HPO4 concentration ([P] = 17.5 µM). Glassware containing P-poor medium was washed previously with HCl (10%) to eliminate P traces. Light experiments were carried out using the original BG11 medium under different continuous light intensities: low light (LL) conditions consisted in a 10 μmol photons m−2s−1 irradiance and were used as control for the comparison of oligopeptide compositions, while medium (LM) and high light (LH) conditions provided 150 and 400 μmol photons m−2s−1, respectively. Sterile glassware with treatment-specific nutrient medium was inoculated with 1 mL aliquots of the original cultures in the exponential phase and placed under the appropriate light conditions. The resulting cultures were maintained under the same temperature, and their growth was monitored by spectrophotometric readings at 750 nm. Culture growth rates were calculated for each treatment during the exponential phase [μ = ln(Nt/No)/Δt]. Nutrient treatments were cultured under continuous light irradiance of 70 μmol photons m3 or 5 μL of free cell suspension were collected with disposable glass capillaries and placed in 0.2 mL Eppendorf tubes, which were allowed to dry at room temperature during 2 h. Ten microliters of an acetonitrile, ethanol and water (1:1:1) extractant solution, acidified with 0.03% (v/v) trifluoroacetic acid (TFA), were then added to each sample. Samples were introduced into liquid nitrogen for 2 h to induce cell lysis and stored at −80 °C until analyzed. For MS analysis, 0.5 μL of individual samples analyte solution were placed onto the matrix spots of a Prespotted AnchorChip , which contain α-cyano-4-hydroxycinnamic acid (HCCA) as co-crystallizing matrix. Spots were allowed to dry at room temperature and were washed with ammonium phosphate (10 mM) for 5 s before MALDI-TOF analysis. Mass spectrometric analysis was performed on a Bruker Reflex MALDI mass spectrometer equipped with a time of flight (TOF) mass analyzer on positive ion detection mode and reflector mode. Acceleration voltage was set to 25 kV and mirror voltage to 26.3 kV. Mass spectra were accumulated from 1000 laser pulses scanning the entire sample spot for a mass range of 500–2000 Da.During the active growth phase and until the stationary growth phase was achieved, individual colonies or free cells were collected for the determination of their oligopeptide compositions through MALDI-TOF MS. Twenty replicates for each strain-treatment-time combination were analyzed. Colony biovolume  and optical density (for unicellular cultures) were correlated to cell numbers for each strain (data not shown), were used to indirectly ensure sufficient biomass in the samples (>6000 cells) and, thereby, obtain unambiguous mass spectra. Single colonies of at least 0.03 mmk = 2), so that data variability within the dataset was separated into two clusters. The resulting clusters contained samples with similar  peptide compositions.The resulting mass spectra were processed using Bruker FlexAnalysis software (version 3.0). The SNAP algorithm  was used for mass determination using a signal to noise threshold of 6. Putative oligopeptides were identified according to their mass/charge ratio, compared with previously described oligopeptides . The occTo evaluate whether applied treatments caused changes in the peptide composition compared to controls, the percentage of treatment and control replicates assigned to each cluster was examined. When treatments induced variations in the peptide fingerprint, control replicates were majorly and selectively concentrated in cluster A, while most of the treatment replicates were grouped in cluster B. Conversely, when assayed conditions did not cause changes in the peptide composition, non-selective assignment occurred, having samples corresponding to both control and treatment majorly assigned to the same cluster.i.e., those showing IDFs below 60%). RIT values represent the delimitation, in terms of mass spectral relative intensity, between stable and unstable peptides for each treatment and strain.Given that chemotypical subpopulations are delimited by their respective peptide compositions, which are usually defined by the presence of individual oligopeptides, the gain or loss of one or more peptides would imply a change in the chemotype-specific fingerprint. Conserved oligopeptide patterns were, hence, considered those which conserved their complete oligopeptide combination regardless of treatments and time. Therefore, stability was analyzed as the consistency in detection of individual peptides. Detection frequencies were expressed as a percentage of positive detections among same-treatment replicates  and aim to provide a metric of the variations in detection of individual peptides between replicates. Oligopeptides presenting IDFs below 60% were regarded as unstable signals and consequently considered to account for changes in the oligopeptide fingerprint. This value aimed to provide a conservative limit to discern between stable and unstable peptides. To express the extent of the fingerprint instability, a relative intensity threshold (RIT) was calculated, which represents the maximal mean relative intensity among unstable oligopeptides (The stability of oligopeptide fingerprints used for the definition of cyanobacterial chemotypes has not been assessed. Our results evidence that oligopeptide fingerprints may be subject to distortion under suboptimal physiological conditions, consisting in the gradual loss of low intensity signals corresponding to minor microcystin variants. Changes in the oligopeptide patterns may be misinterpreted as shifts in the composition of chemotypes and may lead to overestimation of their diversity in natural populations. Therefore, criteria for the definition of oligopeptide chemotypes should be based on the detection of high intensity producing peptides, which can be consistently detected over the complete range of environmental conditions, allowing an accurate analysis of the dynamics of chemotypes in natural ecosystems.


One–fifth of patients with seemingly ‘curable’ pancreatic ductal adenocarcinoma (PDA) experience an early recurrence and death, receiving no definable benefit from a major operation. Some patients with advanced stage tumors are deemed ‘unresectable’ by conventional staging criteria (e.g. liver metastasis), yet progress slowly. Effective biomarkers that stratify PDA based on biologic behavior are needed. To help researchers sort through the maze of biomarker data, a compendium of ∼2500 published candidate biomarkers in PDA was compiled  p. e1000046).survival tissue microarray (termed s-TMA) comprised of short-term  and long-term survivors  who underwent resection for PDA . The s-TMA functions as a biological filter to identify bona fide prognostic markers associated with survival group extremes . Based on a stringent selection process, 13 putative PDA biomarkers were identified from the public biomarker repository. Candidates were tested against the s-TMA by immunohistochemistry to identify the best markers of tumor biology. In a multivariate model, MUC1  and MSLN  were highly predictive of early cancer-specific death. By comparison, pathologic factors  had ORs below three, and none reached statistical significance. ROC curves were used to compare the four pathologic prognostic features (ROC area = 0.70) to three univariate molecular predictors  of survival group .Building on this compendium, we constructed a MUC1 and MSLN were superior to pathologic features and other putative biomarkers as predicting survival group. Molecular assays comparing cancers from short and long survivors are an effective strategy to screen biomarkers and prioritize candidate cancer genes for diagnostic and therapeutic studies. While pancreatic ductal adenocarcinoma (PDA) is typically aggressive as compared to most other cancers, the disease is comprised of a range of biological phenotypes. Roughly 20% of patients who undergo resection will live at least 5 years, and a similar percentage of patients will recur early after resection and die of disease within a year Pathology reports include basic information regarding the stage and grade of the tumor, and currently provide the best available prognostic information. Conventional pathologic features remain the prognostic gold standard (e.g. lymph node status and histologic grade). However, across multiple large studies, adjusted hazard ratios for pathologic features are below two Improved prognostic information is a priority of cancer research. First, accurate prognosis informs discussions between oncologists and patients about the natural history of pancreatic cancer. Second, the information can guide treatment decisions with implications for both quality of life and cancer-related outcomes. The most biologically aggressive PDAs (such as those that recur soon after resection) are best treated initially with systemic therapy, as opposed to major surgery. Pancreatic surgery delays systemic treatment by a minimum of 2 months and exposes the patient to substantial operative risk with little expected benefit. On the other hand, patients with indolent cancers with oligometastatic disease may benefit from an aggressive surgical approach, as has become standard of care in selected patients with metastatic colorectal cancer High impact studies based on hundreds of patient samples have improved prognostic capabilities in multiple cancer types  In the present study, we used immunohistochemistry to interrogate a dichotomous set of resected PDAs (n = 137) comprised exclusively of aggressive  and relatively less aggressive  cancers, for true predictors of survival. A panel of 13 promising PDA biomarkers was selected from literally thousands of published PDA candidate biomarkers using a rigorous selection strategy (described in detail below), from on a public compendium of PDA biomarkers  [22]. UsThis study was approved by the Memorial Sloan-Kettering Cancer Center (MSKCC) institution review board. Patients were included if they underwent a pancreatic resection for invasive tubular type  ductal adenocarcinoma (PDA) after the year 2000, and either died of disease within 1 year of resection  or survived at least 30 months . The specific survival boundaries were chosen for two reasons: first, to yield groups that were sufficiently powered for the analysis, yet had comparable sample sizes to each other; second, so that that the time interval between the two survival groups (at least 1.5 years in the present study) emphasized tumor biology over treatment related determinants of survival. For instance, adjuvant treatment provides a survival benefit of roughly 3 months for PDA Clinicopathologic information was extracted from the institutional pancreatic tumor database and from electronic patient records. Relevant clinical variables included postoperative chemotherapy, radiation therapy, and patient survival. Pathologic data included lymph node status (positive vs. negative), tumor differentiation (poor vs. moderate/well), size (≥3 cm vs. <3 cm), and resection margin status (positive vs. negative). Microscopic disease at the pancreatic neck, bile duct, duodenum, and uncinate margins were categorized as positive.The TMA was constructed from tissue cores obtained from formalin-fixed, paraffin embedded tissue blocks in 151 patient samples. In all cases, tissue samples were derived from resected primary ductal adenocarcinomas of the pancreas. The technician placed the samples on the TMA in a blinded fashion ensuring that IHC interpretation by the study investigators was unbiased. The TMA was constructed as follows: a representative block of tumor was obtained and a corresponding H & E stained slide was examined under a microscope for foci of high neoplastic cellularity. Triplicate cores were taken from the index blocks and transferred to a virgin block for TMA processing with an automated tissue array machine . TMA sections were then cut from the block in preparation for immunohistochemistry experiments.number of independently published IHC studies cited in the central biomarker repository. Using this strategy, 13 PDA biomarkers were identified as the subject of four or more IHC-based peer-reviewed studies, and additionally were already optimized in our pathology core . The first 10 were selected for testing in the present study. In addition, three antibodies were included as representative samples from the remaining choices of putative PDA biomarkers: CEACAM6 (2 previous IHC-based publications), MYC (2 publications), and MUC2 (1 publication). MUC2 was believed to be particularly intriguing due to its association with indolent pancreatic tumors, in direct contrast to MUC1, which has been linked with more aggressive pancreatic tumor subtypes A published compendium of putative pancreatic cancer biomarkers based on a comprehensive literature search lists 2,516 overexpressed genes (∼10% of the genome) in pancreatic cancer Immunohistochemical analyses were performed by a standard streptavidin-biotin-peroxidase procedure. Labeled TMA sections were subjected to heat-induced epitope retrieval with the Ventana Discovery XT automated system . Primary antibodies and their dilutions included: BCL2 , CASP3 (1∶300), CCND1 , CEACAM6 , EGFR , ERBB2 , MSLN , MUC1  Immunohistochemical review was performed by an expert pancreatic pathologist (L.H.T.) and recorded by a different study investigator (J.M.W). SMAD4, MUC2, and BCL2 were scored as negative or positive by IHC based on previous scoring strategies The analysis was performed using Intercooled Stata 8.2. Categorical variables were tested by the Fisher’s exact test, continuous variables by the Wilcoxon rank sum test, and standard logistic regression was performed for multivariate testing. Continuous variables were tested using the rank sum test. A comparison of multivariate regression models was performed to identify the best prognostic model using receiver operating characteristic analysis and the associated Harrel’s C-index . In the present analysis, the C-index measures how well a particular multivariate model of predictors discriminates between short- and long-term survival groups. The values ranged between 0 and 1. A value of 0.5 indicates no predictive ability (random prediction) and appears as a diagonal line on an ROC graph, whereas values above 0.5 indicate good predictability, and appear as curvilinear plots above the diagonal. When two ROC curves do not intersect, the one with a higher C-index dominates over the other. All statistics were two-tailed with a p value <0.05 indicating statistical significance.There were 79 (58%) patients in the long-term survivor group and 58 (42%) in the short-term survivor group. Conventional pathologic features  and patient variables  were analyzed as predictors of survival group . None ofThe prognostic accuracy of three different models, as estimated by the Harrell’s C-index, was compared, graphed and tabulated . The modExpression patterns of the 13 candidate prognostic markers . In the total cohort, 15% of patients had an IHC score of 0, 23% had 1+, 33% had 2+, and 30% had 3+. The proportions of patients that were in the short survivor group at each separate IHC score increased in a linear fashion . Specifically, 5% were in the short survival group with an IHC score of 0, 29% with 1+, 49% with 2+, and 68% with 3+ . The negAs compared to MUC1 expression, the pattern of MSLN expression in the total cohort was slightly weighted towards lower IHC scores: 63% of patients had 2+ or 3+ MUC1 labeling while 48% had comparable MSLN labeling (p = 0.02). However, like MUC1, there was a strong association between MSLN expression and early cancer-specific mortality (p<0.0001). Again, a linear relationship was observed between the IHC score and the proportion of patients in the poor survival group . In the different IHC score categories, the percentage of patients that were in the short survival group were as follows: 20% of the patients with an IHC score of 0, 35% with 1+, 49% with 2+, and 81% with 3+ .MUC2 expression was associated with long survival in contrast to MUC1 and MSLN (p = 0.03). MUC2 expression was uncommon overall (15%) in PDA. Short-term survivors expressed MUC2 in just 7% of cases. Long-term survivors expressed MUC2 in a greater proportion, although expression was still uncommon (22% of cases).A multivariate logistic regression analysis was performed which included significant univariate biomarker predictors of survival  as well as the four commonly reported pathologic features . MUC1 anEarly cancer recurrence and mortality after pancreatic resection (within one year) remain disheartening experiences for clinicians. In these instances, patients with seemingly “resectable” disease have major resections with “curative intent,” yet do not receive any definable benefit, occasionally at the cost of significant morbidity or even mortality. Our institutional data suggests this scenario occurs in one–fifth of patients who undergo pancreatic resection for PDA Studies designed to identify reliable prognostic markers face two particular challenges. First, extrinsic determinants of survival which are independent of a tumor’s molecular profile confound biomarker analyses. Consider a scenario in which the difference in overall survival between two patients after pancreatic resection is only 3 months. The survival difference may be related to patient performance status, social factors, medical comorbidities, chemotherapy response, treatment toxicity, surgical complications, or a number of other possibilities. Each of these factors may minimally contribute to patient survival, and would require a study with very large statistical power to fully characterize each one. Furthermore, these survival factors are not typically associated with biomarker expression patterns (chemotherapy response and toxicity may be exceptions). The present study minimizes noise from alternative and less significant survival factors by excluding patients with intermediate survival (12–30 months). Except for rare instances, tumor biology would be expected to be the principal driver of survival groups defined by a time gap of this magnitude (a minimum of 1.5 years separates short and long survivor groups).survival TMA may be better suited for biomarker discovery investigations in PDA, because it emphasizes tumor biology. The sample size in the present study compares favorably with other TMA studies (top quartile). Most important, this study likely includes the largest number of patients at the survival extremes.We identified 20 other studies in the literature that analyzed protein biomarkers using TMAs of PDA . Unlike The second challenge for biomarker surveys of PDA with IHC is to devise a rational strategy to select the best molecular candidates for study. There are roughly 30,000 human proteins, and 10% have been reported as overexpressed in PDA Biomarker selection in the present study was based on the recently published and centralized biomarker repository for PDA IHC analysis revealed that 10 of the13 candidates were non-informative as prognostic markers in this study cohort. These negative observations provide convincing evidence  that this group of putative pancreatic cancer biomarkers are clinically irrelevant for prognosis. In the univariate analysis, MUC1 and MSLN expression were associated with aggressive cancer biology  and MUC2 expression was associated with favorable biology . Only MUC1 and MSLN were robust prognostic factors in the multivariate model, adjusting for conventional pathologic features . DiffuseThis study validates MUC1 and MSLN as biomarkers of aggressive pancreatic cancer biology. The implications of these findings must be interpreted in the context of the study design, and the role of these proteins as prognostic markers in the clinical management of PDA remains uncertain. While each oncoprotein has been the focus of over 200 studies in PDA, there are no large-scale studies of tumor samples that have thoroughly examined them as prognostic biomarkers using a comparable graded IHC scoring system. Some previous studies have observed survival differences associated with high and low expressing tumors (MUC1 or MSLN), but are limited by small sample sizes, the absence of tiered IHC scoring systems, and unadjusted statistics Certain biomarkers included in the study had expression patterns that differed from previous reports. For instance, MSLN expression (1+ or greater) was observed in 71% of patients with PDA in the present study  holds great promise as a serum and pleural fluid marker for malignant pleural mesothelioma We are presently evaluating additional candidate prognostic markers against the s-TMA to further optimize the predictive model. Based on the strategy used to select candidate markers in the present study, additional intriguing proteins include MUC5AC, BIRC5, and ITGB4. A high-throughput proteomic or transcriptomic analysis of survival extremes could identify novel prognostic markers, but is best suited for tumor samples enriched for neoplastic cells such as tumor cell lines or xenografts (as opposed to primary tumor tissue such as the samples in this study with abundant stroma) This study presents the results of a survival-based TMA (s-TMA), comprised of patient tumor samples associated with short and long-term survival after resection for PDA. The s-TMA was used to identify bona fide protein markers of aggressive tumor biology. MSLN and MUC1 were highly significant predictors of early cancer-specific mortality, and were superior to conventional pathologic features as prognostic markers.


By transferring such model-derived trained (donor) animal hippocampal firing patterns via stimulation to coupled naïve recipient animals, their task performance was facilitated in a direct “donor-recipient” manner. This provides the basis for utilizing extracted appropriate neural information from one brain to induce, recover, or enhance memory related processing in the brain of another subject.The critical role of the mammalian hippocampus in the formation, translation and retrieval of memory has been documented over many decades. There are many theories of how the hippocampus operates to encode events and a precise mechanism was recently identified in rats performing a short-term memory task which demonstrated that successful information encoding was promoted via specific patterns of activity generated within ensembles of hippocampal neurons. In the study presented here, these “representations” were extracted via a customized non-linear multi-input multi-output (MIMO) mathematical model which allowed prediction of successful performance on specific trials within the testing session. A unique feature of this characterization was demonstrated when successful information encoding patterns were derived online from well-trained “ To understand the neural basis of memory, several features of the context in which the memories occur and are utilized, and the functional aspects of the brain areas involved, need to be identified and controlled  task. These findings confirm the functional significance of a previously identified “hippocampal prosthesis”  electrodes implanted bilaterally in the hippocampus in each hemisphere to provide single neuron firing data from 8 pairs of aligned CA3-CA1 probes arranged at 200 μm intervals along the longitudinal axis in the dorsal hippocampus in rodent brain Figure . The neuSupporting Material) allowed prediction of CA1 neuron firing “output” patterns based on the “input” to the model (CA3 neuron firing) using Laguerre expansions of Volterra Kernels to determine the temporal relationships between spike occurrences recorded in these two areas during the task . Since both the pattern and time of application of the strong code CA1 SR stimulation were related directly to the MIMO model detection of corresponding CA3 input firing, it was also possible to deliver CA1 SR stimulation on trials in which “strong code” input patterns were not detected in CA3 recordings. This provided the means to facilitate performance above control levels by delivering strong code CA1 SR stimulation on trials that normally did not generate strong codes in CA3 or CA1 naturally  a well-trained “donor rat” performed the DNMS task in one chamber at the same time as (2) a delay-naïve “recipient rat” was tested at the same time in a different chamber in a trial synchronized manner  in order to correctly select the opposite lever in the Non-match phase  delivered on half the trials from synchronously performing donor rats. Donor rat SR CA1 stimulation allowed recipient rats to perform significantly better than if they did not receive stimulation (No stim) and these levels are compared with their higher performance on trials in which no delay was interspersed (0.0 s). The overall performance of all recipient is shown in Figure n = 23).The employment of donor rat stimulation was further verified by the fact that performance was facilitated in a delay dependent manner  on both left and right SR lever trials. Although there were slight differences with respect to the spatiotemporal delivery of CA1 stimulation pulses, the overall patterns related to the time of execution of the SR were highly similar. The graphs to the right in Figure recipient rat on similar types of trials with stimulation and non-stimulated extended delays from the same two donor rats. It is clear that stimulation generated by both donor rats on different trials in the same session facilitated performance of the recipient rat in nearly identical fashion. Figure recipient rats (n = 5) over the 3 delay intervals for all donor rat (n = 6) stimulation (Stim) vs. non-stimulated (Non-Stim) trials compared to trials in which no delay was imposed (0 s).The physiologically specific nature of the  Figures  in the sdonor rat SR stimulation was the basis for the improved performance of recipient rats included: (1) changing the donor rat stimulation patterns in different ways such as scrambling coefficients  after the SR . None of the latter control procedures produced significant increases in recipient rat performance above that exhibited on trials with the same delays and no donor rat stimulation  delR Figure , and (3)n Figure .donor as well as delay-naïve recipient rats, is the actual neural basis for the enhancement invoked by delivery of MIMO SR CA1 stimulation and also the type of changes that occur under normal conditions related to improved vs. impaired performance in the same subjects. The influence of administered MIMO CA1 stimulation on synaptic connectivity during the session was assessed using local field potentials (LFPs) recorded from each of the 8 CA1 locations generated by stimulating a single electrode location in CA3 on the same array  in 10 ms segments as shown in Figure diff waveforms reflect changes in particular components of the LFP generated from the same CA3 stimulation location related to both excitatory and inhibitory input over the same range of voltages, before and after behavioral sessions in which SR stimulation was, or was not, delivered.A major factor that relates to the above demonstration of enhanced memory in the y Figure  with miny Figure . CA1 LFPdiff LFP waveforms for a single trained animal following SR stimulation (blue) vs. non-stimulation (red) sessions. It is clear that the LFP changes were related directly to both excitatory (40–80 ms) and inhibitory (90–175 ms) components of well-characterized LFPs recorded from the cell layer at each of the eight CA1 locations  vs. SR stimulation (blue curve) sessions in Figure diff values in terms of subtracted Stim session Post-Prediff LFP waveforms from similar Non-stim session waveforms, i.e., Diffstim—Diffnon−stim LFP values. Thus, the dotted curve in Figure diff LFP difference curves (red and blue) but using Post-Prediff LFPs instead to provide the resulting dotted curve average difference of LFP waveform for SR stim vs. non-stim sessions . Since this average difference (Diffstim—Diffnon−stim) across all animals (n = 5) reflects alteration in the same LFP components as shown for individual waveforms in a single animal (red and blue Post-Prediff LFPs), it is clear that synaptic processes mediating CA3-to-CA1 transmission were increased by MIMO stimulation delivery during the DNMS sessions in which performance within and between animals was facilitated  immediately enhance task-specific performance, (2) repair damaged or impaired task-dependent brain circuitry, and possibly even, (3) provide neural encoding of task-relevant information without prior training. The long history of investigation with hippocampal recording in the behavioral context employed, and prior collaboration perfecting application of the MIMO model to these recordings (Hampson et al., donor/recipient memory transference could occur as demonstrated here. However, the fact that transferred patterns of electrical brain stimulation have significant functional impact and are capable of modifying performance via strategic online delivery provides another demonstration of donor-recipient brain compatibility (Pais-Viera et al., donor/recipient type devices (Jarosiewicz et al., These results provide important insight for extending The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recently, we have shown that connexin (Cx) 30 hemichannels are localized to the non-junctional apical membrane of cells in the distal nephron-collecting duct (CD) and release ATP into the tubular fluid upon mechanical stimuli, leading to reduced salt and water reabsorption. Cx30−/− mice show salt-dependent elevations in BP and impaired pressure-natriuresis. Thus, we hypothesized that increased tubular flow rate leads to Cx30-dependent purinergic intracellular calcium ([Ca2+]i) signaling in the CD. Cortical CDs (CCDs) from wild type and Cx30−/− mice were freshly dissected and microperfused in vitro. Using confocal fluorescence imaging and the calcium-sensitive fluorophore pair Fluo-4 and Fura Red, we found that increasing tubular flow rate from 2 to 20 nl/min caused a significant 2.1-fold elevation in [Ca2+]i in wild type CCDs. This response was blunted in Cx30−/− CCDs . To further test our hypothesis we performed CD [Ca2+]i imaging in intact mouse kidneys in vivo using multiphoton microscopy and micropuncture delivery of the calcium-sensitive fluorophore Rhod-2. We found intrinsic, spontaneous [Ca2+]i oscillations in free-flowing CDs of wild type but not Cx30−/− mice. The [Ca2+]i oscillations were sensitive also to P2-receptor inhibition by suramin. Taken together, these data confirm that mechanosensitive Cx30 hemichannels mediate tubular ATP release and purinergic calcium signaling in the CD which mechanism plays an important role in the regulation of CD salt and water reabsorption.ATP in the renal tubular fluid is an important regulator of salt and water reabsorption via purinergic calcium signaling that involves the P2Y Renal epithelial i , whereas no significant changes were detected in CCDs from Cx30−/− mice   form of fluorophores for confocal fluorescence imaging. The calcium-sensitive fluorophore Rhod-2 was employed in the present experiments, as red fluorescent probes are preferred over green dyes due to the low overlap of their emission with the significant renal tissue autofluorescence. Initially, we used the multicell bolus loading (MCBL) technique that we previously described for real-time imaging of intracellular pH and [Ca2+]i in the proximal tubules . In kidneys from wild-type mice a subset of CD cells showed spontaneous oscillations in Rhod-2 fluorescence reflecting changes in [Ca2+]i .To further support our hypothesis that flow-induced [Cai Figure . In cont Figures . To testin vitro microperfused CCDs and ratiometric calcium imaging we first found that the classic phenomenon of tubular flow-induced increases in CD [Ca2+]i was dependent on the expression of Cx30. In addition, spontaneous [Ca2+]i oscillations in free-flowing CDs were observed in the intact living kidney of wild type but not Cx30−/− mice and the [Ca2+]i responses were found to be sensitive to suramin, a P2 purinergic receptor inhibitor. Our results are consistent with the presence of mechanosensitive, ATP releasing Cx30 hemichannels in the distal nephron-CD system and established that CD cell [Ca2+]i responses are an integral element of the intra-tubular autocrine/paracrine purinergic system in the CD that is driven by Cx30-mediated ATP release. The present studies also established the exciting new technical advance of directly and quantitatively visualizing CD [Ca2+]i responses in the intact mouse kidney in vivo.This study is a direct and logical continuation of our recent line of research which aims to characterize ATP releasing mechanisms and their physiological relevance in the kidney. In the present experiments we used confocal fluorescence imaging approaches to further examine the function of one particular ATP conduit, the Cx30 hemichannel in the renal CD. Using 2+]i responses were measured as an indicator of ATP release .The present findings are in line with our previous study showing that Cx30-mediated ATP release in the CD can be triggered by mechanical forces, for example by increases in tubular fluid flow rate  that are known to exist due to renal hemodynamic feedback mechanisms  since in the in vitro dissected and microperfused CD, in which fluorophore loading was very efficient, all cells produced flow-induced [Ca2+]i responses . However, these important questions need to be addressed in future work using more suitable experimental approaches .It should be noted that the present studies detected s Figure . Also, i2+]i signaling could be regulating epithelial transport processes in other tubular segments in other species. In addition, the ATP channel pannexin 1 which was recently localized to the apical plasma membrane of several renal tubule segments including the CD (Hanner et al., These studies were carried out in mice, where the expression of Cx30 is restricted to the intercalated cells of the CNT and CCD (McCulloch et al., 2+]i responses in the CD in the intact kidney in vivo and further characterize the function of ATP releasing Cx30 hemichannels in the mouse CD. Our results established that Cx30-dependent [Ca2+]i responses are an integral element of the intra-tubular autocrine/paracrine purinergic system in the CD that plays an important role in the regulation of renal salt and water reabsorption, the maintenance of body fluid and electrolyte balance and normal blood pressure.In summary, the present studies successfully visualized [CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CCL24) which was consistently transcribed higher amongst MDD patients relative to controls and BPD patients, and C-C chemokine receptor type 6 (CCR6) which was consistently more lowly transcribed amongst MDD patients relative to controls. Results detailed here provide preliminary evidence that transcriptional measures within inflammation-related genes might be useful in aiding clinical diagnostic decision-making processes. Future research should aim to replicate findings detailed in this exploratory study in a larger medication-free sample and examine whether identified biomarkers could be used prospectively to aid clinical diagnosis.Mood disorders consist of two etiologically related, but distinctly treated illnesses, major depressive disorder (MDD) and bipolar disorder (BPD). These disorders share similarities in their clinical presentation, and thus show high rates of misdiagnosis. Recent research has revealed significant transcriptional differences within the inflammatory cytokine pathway between MDD patients and controls, and between BPD patients and controls, suggesting this pathway may possess important biomarker properties. This exploratory study attempts to identify disorder-specific transcriptional biomarkers within the inflammatory cytokine pathway, which can distinguish between control subjects, MDD patients and BPD patients. This is achieved using RNA extracted from subject blood and applying synthesized complementary DNA to quantitative PCR arrays containing primers for 87 inflammation-related genes. Initially, we use ANOVA to test for transcriptional differences in a ‘discovery cohort’  and then we use t-tests to assess the reliability of any identified transcriptional differences in a ‘validation cohort’ . The two most robust and reliable biomarkers identified across both the discovery and validation cohort were Chemokine (C-C motif) ligand 24 ( The term ‘mood disorder’ refers to a category of psychiatric illness that is characterized by a pathological distortion of affect Despite the establishment of clinical diagnostic criteria for MDD and BPD, the heterogeneous nature of these disorders, the similarities they share in their clinical presentation, and the absence of specific biomarkers, means there are relatively high rates of misdiagnosis The clear need for an objective, empirical method of diagnosis has led to genome-wide association studies (GWASs) attempting to identify genes associated with MDD and BPD. However, despite twin studies suggesting mood disorders are moderately heritable, GWASs have largely been unsuccessful in identifying genes robustly associated with MDD Cytokines are a group of cell-signaling proteins which, in the periphery, aid inflammatory processes and the immune system to form coordinated responses to infection The current study aims to identify transcriptomic biomarkers in the inflammatory cytokine pathway which could be used to distinguish between controls subjects, MDD patients and BPD patients. We achieve this using RNA extracted from whole blood and examine an extensive set of inflammatory-related transcripts including genes coding for: interleukins and interleukin receptors, chemokines and chemokine receptors, the tumor necrosis factor cytokine family and receptors, and other inflammatory regulators. We initially test for differences in a discovery cohort , and then attempt to replicate any findings from our discovery cohort in a validation cohort .Patient samples used in this study were collected from two methodologically similar studies, the Bipolar Association Case–Control Study (BACCS) BPD patients in BACCS were recruited from three sites, Toronto Canada, London UK and Dundee UK. BACCS was a community-based study, where subjects were recruited from psychiatric clinics, hospitals, primary care physicians and patient support groups. BPD patients were included in the study if they were over the age of 18 and had been diagnosed with Bipolar I or Bipolar II disorder as defined by the DSM-IV or ICD-10 The current study utilized 40 BPD patient samples in total  collected only from the Dundee UK site, as this was the only site to collect blood for transcriptomic experiments. The subset used here was randomly selected from a larger group of samples. Further patient characteristics are detailed in MDD patient samples were collected as part of the European study GENDEP, which is a 12-week partially randomized open label pharmacogenetic study. Patients were selected if they were diagnosed with MDD of at least moderate severity according to ICD-10 or DSM-IV criteria ABCF1) The current study utilizes 45 patient samples in total , which were randomly selected from the larger GENDEP sample set. Blood samples were collected both at the start of GENDEP and after eight weeks of treatment with escitalopram as described previously We also utilized blood which was collected at the start of GENDEP to ascertain how stable transcriptional biomarkers were at differentiating MDD patients from other subject groups. At the start of GENDEP all patients had been drug-free for two weeks and were all in a clinically significant mood state (BDI>10). We assess whether transcripts identified in the discovery cohort and replicated in the validation cohort, continue to differentiate MDD patients from other subject groups when blood is collected at a different time point, during a different mood state, and during the absence of medication. and Control subjects were selected from BACCS where they were screened for lifetime absence of psychiatric disorders using a modified version of the Past History Schedule The BACC and GENDEP studies were approved by The Joint South London and Maudsley NHS Trust Institute of Psychiatry Research Ethics Committee and at each participating centre and all subjects provided written informed consent.All blood samples from BACCS and GENDEP were collected in 10 ml PAXgene tubes  and stored at −80°C. Prior to the start of gene expression studies, PAXgene tubes were allowed to thaw for 12 hours at room temperature. RNA extraction was performed using the Qiagen PAXgene Blood miRNA Kit  following the standard manufacturer's protocol. The purity and quantity of RNA was measured using the Nanodrop, ND1000 . All samples had 260/280 ratios of between 1.9 and 2.3. RNA integrity numbers (RINs) were furthermore assessed using the Agilent 2100 Bioanalyzer  and the average RIN was 8±1.5.2 HT First Strand Kit following the manufacturer's protocol. Briefly, following genomic DNA removal, the samples were incubated for 15 minutes at 42°C with 6 µl of BC4 RT Mastermix (SABiosciences). The reverse transcriptase enzyme was subsequently inactivated at 95°C for 5 minutes. cDNA samples generated were stored at −20°C prior to use in the qPCR experiments.Reagents used in the quantitative PCR (qPCR) component of the study were manufactured by SABiosciences . Complementary DNA (cDNA) was prepared using 1 µg of total RNA and the SABiosciences RTIL11), interleukin-6 (IL6) and the glucocorticoid receptor (NR3C1). Each array contained five housekeeping genes for normalization. These include: β2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase (HPRT1), ribosomal protein L13a (RPL13A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB). The three most stable housekeeping genes were selected based on RefFinder analyses and used for normalization across samples.Customized 384-well arrays were designed for qPCR experiments. These arrays contained lyophilized primers for the 84 genes listed in the commercially available Human Inflammatory Cytokines & Receptors PCR Array (SABiosciences), with the addition of gene primers for interleukin 11 (2 qPCR Master Mix (SYBR green), 102 µl of diluted synthesized cDNA and 448 µl RNAse free water, with a total volume of 1100 µl for each sample. Each qPCR array contained the following controls: human genomic DNA control (gDNA), reverse transcription control (RTC) and a positive PCR control (PPC). To ascertain whether samples passed quality control checks for gDNA and RTC, the manufacturer's quality control criteria were applied. The qPCR reactions were performed using the ABI Prism 7900HT Sequence Detection System . Thermal cycling conditions consisted of an enzyme activation stage (95°C for 10 minutes), followed by 40 cycles of a denaturation stage (95°C for 15 secs) and a hybridization and extension stage (65°C for 1 minute). The software program SDS 2.3 (Applied Biosystems) generated cycle threshold values (Ct) from the data collected, see Table S1 for raw Ct values.Each 384-well array was designed to analyze four samples simultaneously. The qPCR reagents used consisted of: 550 µl of 2X SABiosciences RTt values of greater than 37 were removed and excluded from further analysis as such high Ct values are indicative of very low expression levels. Furthermore, if as a result of data removal, a transcript showed missing data for more than half of the total patient sample, that transcript was excluded from further analysis. The relative expression of target genes was calculated by subtracting the mean Ct of the selected reference genes from the Ct of the target gene to generate ΔCt values http://www.leonxie.com/referencegene.php) t were used in statistical calculations and adjusted 2t−ΔC were used to generate plots Cp2) was calculated as an estimate of effect size, by dividing the sum of squares between groups by the total sum of squares. Games-Howell post-hoc tests were subsequently performed to correct for multiple testing and to generate pair-wise comparisons between subject groups d was generated as our estimate of effect size, by calculating the mean differences between our two subject groups, and dividing this result by the square root of the within-groups mean square. Small (d≈0.2), medium (d≈0.5) and large effect sizes (d≥0.8) were then assumed, as according to Cohen d was generated as our estimate of effect size by multiplying the t-test statistic value by two and dividing the result by the square root of the degrees of freedom To ascertain whether significant transcriptional differences existed between control, MDD and BPD subject groups in our discovery cohort, we performed analysis of variance (ANOVA) tests. Partial eta squared , under different conditions . As before, we attempted to validate biomarkers by performing one-tail independent samples t-tests.http://www.R-project.org).All statistical analyses were performed using SPSS Version 15 . Graphs were generated using the ‘plot’ function in R  = 6.438, p = 0.002, ηp2 = 0.134] and interleukin-8 , see Table S3 for full ANOVA results. Games-Howell post hoc analyses were subsequently performed on all genes present on the array. These tests correct for the effects of multiple testing, and generate pairwise comparisons, see Table S4 for full results. ANOVA revealed 11 genes which showed nominally significant transcriptional differences (p≤0.05) between our three subject groups. The most significant differences between subject groups were found in Chemokine (C-C motif) ligand 24 [CCL24, the most significant difference revealed between subjects in our discovery cohort, again significantly differentiated MDD patients from controls  and BPD patients  in the validation cohort, see CCR6 also continued to differentiate MDD patients from controls in our validation cohort , see One-tailed independent t-tests were used in our validation cohort to test whether we could replicate potential biomarkers identified from our discovery cohort, see CCL24 and CCR6 transcription continued to differentiate MDD patients from our other subject groups when blood was collected from a different time point . Again, we found that higher transcription of CCL24 significantly differentiated MDD patients from controls in our discovery cohort  and validation cohort  when MDD blood was collected from a different time point. Similarly, we found that higher transcription of CCL24 significantly differentiated MDD patients from BPD patients in both our discovery cohort  and validation cohort . Additionally, lower transcription of CCR6 continued to differentiate MDD patients from controls in both our discovery cohort  and validation cohort .We additionally used one-tailed independent samples t-tests to observe whether Mood disorders are heterogeneous disorders that are diagnosed when patients display a number of clinical characteristics. The absence of a specific and objective diagnostic test has led to relatively high rates of misdiagnosis for mood disorders, particularly between MDD and BPD patients IL8) and the glucocorticoid receptor (NRC31). Previous reports have found lowered levels of IL-8 protein in the blood of MDD patients relative to controls, and within the cerebrospinal fluid of suicide attempters compared to controls IL8 distinguished both types of mood disorder patient (MDD and BPD) from control subjects  in both the discovery and validation studies support the notion that transcription of these genes could strongly differentiate MDD patients from other subject groups, and thus might indeed be useful as biomarkers.In contrast, higher transcription of orts see Figure 2orts see  section,CCL24 and CCR6 code for genes in the chemokine cytokine family. The chemokines are small chemotactic cytokines that facilitate the migration of immune cells (e.g. to a site of infection) CCL24 codes for a chemokine which is chemotactic for resting T lymphocytes, eosinophils, and to a lesser extent neutrophils CCR6 codes for a G-protein coupled receptor present on immature dendritic cells, B cells and memory T cells, and binds macrophage inflammatory protein 3 alpha CCL24 and CCR6 transcripts as potential diagnostic biomarkers.Both As well as gene transcription offering a more objective method of clinical diagnosis, the fact that it is also a continuous measure gives it certain advantages over currently utilized categorical measures. For instance, continuous or dimensional diagnostic measures are believed to be more stable over time, offer a better measure of symptom severity, and be better predictors of comorbidity and chronicity Although results reported here are promising, there are five main limitations to this study. Firstly, this study is an exploratory study, utilizing relatively small sample sizes, and although we use both a discovery and validation cohort, patients were obtained as subsamples from the same studies, so it only offers a pseudo-independent replication. Therefore replication studies are required in a larger independent sample. Secondly, although we considered the effects of different medications on gene expression profiles, all of our patients were medicated. Based on our previous work on the MDD patient sample used here, we can, with some confidence, rule out the confounding effects of escitalopram treatment IL8 and NR3C1 amongst mood disorder patients. However, the lack of replication in our validation cohort suggests that differences in the transcription of these genes may not be reliable enough to be utilized as biomarkers. Instead, this study emphasized the potential importance of chemokines as biomarkers, and specifically it identifies the potential utility of CCL24 and CCR6 transcripts as novel biomarkers differentiating MDD patients from control subjects and BPD patients. Consequently, this study provides preliminary evidence that CCL24 and CCR6 could be used in conjunction with symptom measures to more accurately diagnose MDD from the outset and differentiate MDD patients from non-depressed subjects and BPD patients. Further replication studies are now required in a larger medication-naïve cohort to further validate these findings.Despite its limitations, the current study utilizes well-characterized clinical samples, stringent quality control steps, normalization protocols and statistical analyses. This study supports previous reports of differences in the expression of Figure S1A bar chart showing the results of RefFinder analyses performed on a panel of five housekeeping genes. The gene names of the housekeeping genes are indicated on the x-axis, along with their expression stability score shown on the y-axis. Lower RefFinder stability scores represent more stable reference genes.(DOC)Click here for additional data file.Table S1A table showing raw Ct values generated for our genes of interest in our total subject sample.(XLS)Click here for additional data file.Table S2Relative expression (ΔCt) values for all subjects, adjusted for PPC, age, sex, current mood status and the presence of comorbid disorders .(XLS)Click here for additional data file.Table S3ANOVA results comparing our three subject groups.(DOC)Click here for additional data file.Table S4Games-Howell pairwise post-hoc results.(DOC)Click here for additional data file.