Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. Here we report that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. We also show that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal.Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC).  In asymmetrically dividing cells, both spindle-dependent and spindle-independent cleavage furrow positioning pathways are involved in cytokinesis. Here the authors find that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow and to complete cytokinesis in Drosophila neuroblasts. The mitotic cycle of symmetrically and asymmetrically dividing cells completes with cytokinesis, the process that physically separates two sibling cells, ensuring the proper partitioning of the nuclear and cytoplasmic contents at the end of cell division. The correct placement of the contractile ring is of fundamental importance, since inaccurate positioning can lead to the partitioning of both nuclei into one daughter cell, causing tetraploidy, tumour formation and cancer2Drosophila; Zen-4 in C. elegans) and Tumbleweed 67In most metazoan cells, the positional cues for contractile ring placement and assembly originate from the mitotic spindle in the form of the centralspindlin complexDrosophila neuroblasts utilize a cortical polarity-dependent pathway for contractile ring placement1111C. elegans Q-neuroblast lineageThere is also accumulating evidence for the existence of spindle-independent cleavage furrow positioning mechanisms111213In neuroblasts, also in other cells containing two cytokinesis pathways, the function and relative contribution of each pathway in cytokinesis is unclear. Thus, in order to elucidate the exact role of the spindle-dependent pathway in asymmetrically dividing neuroblasts, a component needs to be identified, acting only in the spindle-dependent but not in the polarity-dependent pathway. Recently, it was shown that the centralspindlin component Pav also localizes in a spindle-independent mannerDrosophila neuroblasts employ two spatially and temporally separable cytokinesis pathways. The polarity-dependent pathway specifically induces asymmetric Myosin localization, shifting the cleavage furrow to the basal position, but is dispensable for cleavage furrow constriction. The spindle-dependent pathway is using the CPC to stabilize the positioning of the basal cleavage furrow and subsequently completes cleavage furrow constriction.Here we use live imaging, super-resolution microscopy, photoconversion and fluorescence recovery after photobleaching (FRAP) experiments to carefully monitor the dynamic localization of the CPC during asymmetric cell division. We find that the CPC component Survivin only acts in the spindle-dependent pathway and plays an important role in stabilizing basal cleavage furrow positioning, MT bundling and cleavage furrow constriction but is dispensable for asymmetric Myosin localization. With Survivin we thus identified a specific spindle-dependent cytokinesis component, not intersecting with the polarity-dependent pathway. The data presented here extend our model, proposing that asymmetrically dividing Drosophila neuroblasts. In symmetrically dividing mammalian cells, the CPC very dynamically relocalizes from chromosomes to spindle midzone microtubules and to the cleavage furrow , Survivin accumulated right at the center of the mitotic spindle, coinciding with the bundling of microtubules (11n=9), the central spindle and with it central spindle-associated Survivin shifted basally so that the ingressing cleavage furrow is perfectly centred on the Survivin spot that marks the central spindle . Photoconverting Survivin at the metaphase midplane with a short pulse of ultraviolet light resulted in an immediate and robust change in emission that could be traced from metaphase onwards until telophase. We found that photoconverted Survivin was localized on separating kinetochores, the central spindle population and in the furrow region (n=20). However, the possibility remained that newly synthesized or cytosolic Survivin relocalized to the cleavage furrow. We tested this using FRAP and bleached Survivin::GFP at the metaphase plate. In most cases, no signal was detectable at the midbody after complete quenching of Survivin::GFP  mutants; see Methods and refs rod mutant neuroblasts treated with colcemid and expressing the spindle marker mCherry::Jupitern=25; n=25; To test whether microtubules are required for Survivin’s accumulation at the contractile ring, we chemically ablated the mitotic spindle using colcemid (see Methods) in neuroblasts devoid of the spindle-assembly checkpoint  from inscuteable Insc; Fig. 4dsurvivin mutants provide a useful tool to explore the function of the spindle-dependent cytokinesis pathway during asymmetric neuroblast division. However, since the CPC is associated with many functions during mitosis, complete loss of function alleles cannot be used to specifically address Survivin’s function in cytokinesis. To circumvent this caveat, we utilized a temperature-sensitive separation-of-function allele of survivin, specifically disrupting CPC localization and function from anaphase onwards but not beforescpo mutant neuroblasts show cytokinesis defects, but it is unclear which aspect of cytokinesis is compromisedThe data presented so far strongly suggest that Survivin is a specific component of the spindle-dependent cytokinesis pathway. Thus, scpoZ2775/Df(3R)5780  on average in 8.4 min 5780  mutant l80 n=11; ; Table 180 n=11; . scpo muon phase . We concscpo mutants have been reported to perturb central spindle formationscpo mutant MTs appeared dispersed (data not shown). Thus, we asked whether the mitotic spindle is involved in cleavage furrow assembly, initiation, progression or completion of furrow constriction. To this end, we removed the mitotic spindle with colcemid in the spindle-assembly checkpoint mutant rod to allow entry into anaphase . We confirmed the previous result that rod mutant neuroblasts treated with colcemid and thus lacking the mitotic spindle entirely, show normal apical clearing of Myosin and initiate cleavage furrow constriction , but failed to reach the cortex in rod and colcemid-treated neuroblasts entirely  activity during anaphase. To this end, we knocked down AurB specifically in neuroblasts, expressing aurB RNAi with the neuroblast-specific worniuGal4 driver scpo and rod and colcemid conditions  showed the same phenotype; constriction was slow, but finally completed  and established intensity profiles (see Methods), allowing us to compare Myosin dynamics between wild-type and etaphase  and 7a. etaphase  and 7b. roblasts  and 7b. ild type  and 7c.  mutants . Interesl cortex . We perfthe cell . Howeverild type .scpo and dlg;;pins mutant neuroblasts and found that in both scpo and dlg;;pins mutants, the furrow position deviates from the wild-type scenario; scpo mutant neuroblasts shifted the furrow towards the basal cortex, whereas dlg;;pins mutant neuroblasts pushed it towards the apical cortex (rod mutant background) does not affect apical Myosin clearingWe independently measured cleavage furrow positioning at the onset of furrowing in wild-type, l cortex . This daDrosophila neuroblasts111213With the identification of the polarity-dependent cleavage furrow-positioning pathway in Drosophila neuroblasts. Using in vivo live cell imaging of Drosophila neuroblasts in intact larval brains, we find that Survivin dynamically relocalizes from the kinetochores to the central spindle, the cleavage furrow and subsequently the midbody10Here we report that the CPC component Survivin is a specific component of the spindle-dependent cytokinesis pathway in asymmetrically dividing scpo, only disrupting Survivin’s function and CPC localization from anaphase onwardsaurB, the kinase entity in the CPC complex, results in a very similar phenotype.The identification of a specific component of the spindle-dependent cytokinesis pathway in neuroblasts allowed us to test the function of this pathway during asymmetric cell division. Using the separation-of-function allele scpo mutants. Furthermore, knockdown of pav still allows cleavage furrow constriction. Similar results were also obtained for the second centralspindlin component, tumbleweed. Since we see very strong spindle organization defects and polyploidy, we do not think that the lack of a strong furrowing phenotype is owing to partial inactivation of the respective gene products, but favour the hypothesis that these proteins are acting redundantly with other CPC targets. For instance, Septins, membrane-associated filamentous GTP-binding proteins implicated in cytokinesisaurBtum-or pav-deficient neuroblasts (data not shown).What are the CPC targets involved in the completion of contractile ring closure? A prime candidate is the centralspindlin complex, since its localization depends on the CPC3031scpo mutants—and also neuroblasts lacking the mitotic spindle completely—the cleavage furrow is shifted towards the basal cortex. It is important to note that this phenotype is different from mutants compromising the polarity-dependent pathway, in which the cleavage furrow is shifted towards the apical neuroblast cortex, mispositioning it in the middle of the cellWe also found that Survivin and the mitotic spindle contribute towards accurate cleavage furrow positioning; in Drosophila Schneider cellsde novo reassembled from soluble dimers through Rho1 induction at the neuroblast furrow region. Alternatively, the polarity-dependent pathway could induce a basal directed Myosin flow, and stabilized through the CPC and downstream effectors at the forming cleavage furrow. Since Survivin reaches the furrow region at the onset of furrowing, this scenario is plausible. Last but not least, in addition to the polarity-dependent Myosin flow, an apical-directed Myosin flow, originating on the basal cortex, could be induced through the CPC and the mitotic spindle. Indeed, in scpo and colcemid-treated neuroblasts, we see a delay in basal Myosin clearing 5780 (ref. survivin gene), dlgm52 S2214 , His2A::mRFP1UAS-ALD-Survivin::EGFP (this work). UAS transgenes were expressed using worGal4 5780 ref.   using In-Fusion technology . The resulting construct was injected into attP40 and VK0005 (Genetic Services).−1, Glutamine 20 mM, Glutathione 0.04 mg ml−1, and 20-Hydroxyecdysone 5 μg ml−1 according to a recently published reportLive imaging samples were prepared according to a recently published protocol−1.Colcemid experiment: shortly before mounting the brains, the drug was added to supplemented S2-media at a final concentration of 5 μg mlLive samples were imaged with an Andor revolution spinning disc confocal system, consisting of a Yokogawa CSU-X1 spinning disk unit and two Andor iXon3 DU-897-BV EMCCD cameras. A 60X/1.4NA oil immersion objective mounted on a Nikon Eclipse Ti microscope was used for most images. Images contain voxel sizes of 0.22 × 0.22 × 0.5 μm .scpo mutants were performed on a Perkin Elmer spinning disc microscope, equipped with two Hamamatsu C9100-50 frame transfer EMCCD cameras and a sealed chamber allowing to image at 29 °C. A × 63, 1.4NA objective, mounted on a Leica DM6000 was used.Temperature shift experiments for survivin::mDendra2 were used. Photoconversion experiments were performed on an Andor Revolution spinning disc system containing the FRAPPA unit (Andor). Several regions of interests (ROIs) were manually chosen in the GFP channel. Survivin at the metaphase plate was irradiated before anaphase onset. Before photoconversion, single Z planes containing ROIs were scanned for ten time points with maximum speed. Subsequently, ROIs were irradiated with the 405 nm laser line . After photoconversion, the entire neuroblast was scanned with a z-step size of 0.65 μm. Photoconverted Survivin-mDendra2 emits red fluorescence. Converted and unconverted mDendra2 emission were merged in Andor IQ2 and converted into Imaris.The 96-h AEL larval brains expressing one to three copies of survivin::GFP and mCherry::Jupiter were used. Several ROIs were manually chosen in the GFP channel. Before FRAP, single Z planes containing ROIs were scanned for ten time points with maximum speed using 300-ms exposure time. Subsequently, the 488-nm laser line was used to irradiate ROIs  two times in short succession (~3 s pause between irradiations). After FRAP, the entire neuroblast was scanned with a z-step size of 0.65 μm.The 96-h AEL larval brains expressing The following primary antibodies were used: rabbit anti-Aurora B , rabbit anti-Survivin , rat anti-αTubulin . Secondary antibodies were from Molecular Probes and the Jackson Immuno Laboratory.For three-dimensional structured illumination microscopy (3D-SIM), the following primary antibodies were used: mouse anti-αTubulin  and rat anti-Miranda . The following secondary antibodies were used: goat anti-mouse-568  and donkey anti-rat Alexa Fluor 647 .4). After fixing, the brains were washed with PBSBT  and then blocked with 1 × PBSBT for 1 h. Primary antibody dilution was prepared in 1 × PBSBT, and brains were incubated overnight or during 2 days at 4 °C. Brains were washed with 1 × PBSBT three times for 30 min each and then incubated with secondary antibodies diluted in 1 × PBSBT at 4 °C, overnight. The next day, brains were washed with 1 × PBST  three times for 30 min each and kept in Vectashield (Vector Laboratories) mounting media at 4 °C. For super resolution, brains were kept in PBS, then mounted in Vectashield shortly before acquisitions.Larval brains were dissected in Schneider’s insect medium and fixed for 20 min in 4% paraformaldehyde in PEM  equipped with 405, 445, 488, 514, 568 and 642 nm solid-state lasers. Images were acquired using a Plan Apo N × 60, 1.42 NA oil immersion objective lens (Olympus) and four liquid-cooled sCMOs cameras . Exciting light was directed through a movable optical grating to generate a fine-striped interference pattern on the sample plane. The pattern was shifted laterally through five phases and three angular rotations of 60° for each z section. Optical z-sections were separated by 0.125 μm. The laser lines 488, 568 and 642 nm were used for 3D-SIM acquisition. Exposure times were typically between 3 and 100 ms, and the power of each laser was adjusted to achieve optimal intensities of between 5,000 and 8,000 counts in a raw image of 15-bit dynamic range at the lowest laser power possible to minimize photobleaching. Multichannel imaging was achieved through sequential acquisition of wavelengths by separate cameras.Raw 3D-SIM images were processed and reconstructed using the DeltaVision OMX SoftWoRx software package .Supplementary Figures 1-5Survivin dynamically relocalizes during mitosis. Wild type larval neuroblast expressing Survivin::GFP  and the cortex marker Sqh::mCherry (white in merged movie on the left). The neuroblast was imaged every 30 s. Time scale is h:mm:ss. The scale bar is 5 μm.Kinetochore associated Survivin splits up into three Survivin pools. Wild type larval neuroblast expressing Survivin::GFP  and the DNA marker His2A::mRFP1 (white in merged movie on the left). The neuroblast was imaged every 20 s. Time scale is h:mm:ss. The scale bar is 5 μm.Survivin is not sufficient to recruit Myosin and to induce ectopic furrowing. Wild type larval neuroblast expressing ALD-Survivin::EGFP (green in merged movie on the left and the single channel movie in the middle) and Sqh::mCherry (white in merged movie on the left and the single channel movie on the right). The neuroblast was imaged every 30 s. Time scale is h:mm:ss. The scale bar is 5 μm.Cleavage furrow formation and ingression in wild type neuroblasts. Wild type larval neuroblast expressing Sqh::GFP  and the spindle marker mCherry::Jupiter (white in merged movie on the left). The neuroblast was imaged every 30 s. Time scale is h:mm:ss. The scale bar is 5 μm.scpo mutant neuroblasts. Representative scpo mutant neuroblast expressing Sqh::GFP  and the spindle marker mCherry::Jupiter (white in merged movie on the left). The neuroblast contains multiple centrosomes. The neuroblast was imaged every 45 s. Time scale is h:mm:ss. The scale bar is 5 μm.Cleavage furrow ingression is compromized in H4.8rod mutant neuroblast treated with colcemid, expressing the cortex marker Sqh::GFP  and mCherry::Jupiter (white in merged movie on the left). Note that the mitotic spindle is completely depolymerized. The neuroblast was imaged every 10 s. Time scale is h:mm:ss. The scale bar is 5 μm.MTs are required to complete cytokinesis. aurB RNAi treated neuroblast, expressing the cortex marker Sqh::GFP  and mCherry::Jupiter . Note that this neuroblasts contains multiple centrosomes. The neuroblast was imaged every 15 s. Time scale is h:mm:ss. The scale bar is 5 μm.AurB is required to complete cytokinesis. Pav dynamically relocalizes to the cleavage furrow. Wild type neuroblast, expressing the centralspindlin marker Pav::GFP  and mCherry::Jupiter . The neuroblast was imaged every 30 s. Time scale is h:mm:ss. The scale bar is 5 μm.scpo mutant neuroblast expressing Pav::GFP  and the spindle marker mCherry::Jupiter (white in merged movie on the left). The neuroblast is already binucleate due to failed cytokinesis in a previous cell cycle. The neuroblast was imaged every 28.3 s. Time scale is h:mm:ss. The scale bar is 5 μm.Pav localization is compromized in scpo mutant neuroblasts. Representative


Prompt treatment of severe sepsis in the Emergency Department reduces deaths, but the role of prehospital fluid resuscitation is unknown. We sought to determine the risk-adjusted association between prehospital fluid administration and hospital mortality among emergency medical services (EMS) patients admitted with severe sepsis.We performed a prospective, observational study of patients hospitalized with severe sepsis on admission among 45,394 adult EMS encounters taken to 15 hospitals from 11/2009 to 12/2010 by a two-tier EMS system in King County, Washington. The region mandated recording of prehospital intravenous catheter and fluid administration in prehospital records, along with detailed demographic, incident, physiologic, and hospital adjustment variables. We determined the effect of prehospital intravenous catheter or fluid versus no catheter or fluid on all-cause mortality using multivariable logistic regression.P =0.02) compared to no prehospital fluid. The odds of hospital mortality were also lower among severe sepsis patients treated with prehospital intravenous catheter alone .Of all encounters, 1,350 met criteria for severe sepsis on admission, of whom 205 (15%) died by hospital discharge, 312 (23%) received prehospital intravenous fluid, 90 (7%) received a prehospital catheter alone and 948 (70%) did not receive catheter or fluid. EMS administered a median prehospital fluid volume of 500 mL (interquartile range (IQR): 200, 1000 mL). In adjusted models, the administration of any prehospital fluid was associated with reduced hospital mortality  contains supplementary material, which is available to authorized users. Prompt recognition and treatment of severe sepsis and septic shock are keystones of optimal care –4. Such We sought to examine if prehospital fluid administration is associated with hospital mortality in adults with severe sepsis. We examined if either prehospital intravenous catheter or fluid is associated with reduced mortality compared to no intravenous catheter or fluid and tested these associations among septic patients with prehospital hypotension.We examined prospectively collected, population-based data from King County, WA, USA, EMS . We studKing County Medic One serves a population of approximately 750,000 persons who reside in urban, suburban and rural areas over approximately 600 square miles. The EMS system responds to medical emergencies in two tiers. The first tier is comprised of 14 EMS agencies, which comprise firefighter emergency medical technicians trained in basic life support (BLS). The second tier is comprised of a single paramedic agency, which is trained in advanced life support including electrocardiogram rhythm interpretation, intravenous and intraosseous line placement, medication administration and endotracheal intubation. The second tier is dispatched to patients with more severe illness according to criteria-based dispatch assessment or first-tier EMS patient evaluation. Patients are transported to one of 15 hospitals.The triage and care of potential sepsis patients by EMS is at the discretion of the responding personnel. There is no formal sepsis protocol; rather the paramedic discusses cases with an online medical control emergency physician to determine treatment (including intravenous fluids) and disposition. Paramedics are required to perform 35 intravenous catheter starts annually to remain eligible for certification. Only lactated ringers was available for intravenous (IV) fluid during the study period and BLS providers cannot start IV catheters or give IV fluid. No antibiotics were administered by EMS.The King County EMS electronic database is drawn from written and electronic medical incident report forms completed by EMS personnel. Prior to study start, we prospectively modified the medical incident report form such that any paramedic documenting an IV catheter start during the study period is also required to record the administration of prehospital fluid (yes/no) and total prehospital volume (mL). The database was auto-populated with prehospital time intervals and geospatial data from the dispatch center. We linked EMS records to the Washington State Comprehensive Hospital Abstract Reporting System (CHARS) database from 2009 to 2011. CHARS is a state-wide database of all hospitalizations, with accurate diagnostic, procedural and discharge data, including present-on-admission indicators . Prehosp2,%), and Glasgow Coma Scale score (GCS) when available. We abstracted an index of illness severity as determined by EMS personnel: ‘life-threatening’ , ‘urgent’ , and ‘non-urgent’. We defined prehospital location as home, nursing home or medical facility, and abstracted additional prehospital procedures, such as electrocardiogram monitoring, delivery of supplemental oxygen, bag-valve mask ventilation, or intubation. We defined primary prehospital diagnostic categories as respiratory, cardiovascular or neurologic, and calculated a validated prehospital critical illness severity score [We defined prehospital resuscitation in three categories: 1) no IV catheter placed and no fluid administered; 2) IV catheter placed but no fluid administered; and 3) IV catheter placed and fluid administered. We evaluated the placement of IV catheter as a separate category as prior data suggest the procedure may impact patient outcomes in the prehospital critically ill , perhapsty score ,19. We dty score , includia priori secondary outcomes including ICU admission and increasing organ failures during hospitalization . We used only organ failures as defined by the Angus sepsis implementation [The primary endpoint was hospital mortality, defined by disposition in the hospital discharge data. We studied additional entation .Missing data in the King County MedicOne database was variable, for example, ranging from 0% for age to 45% for pulse oximetry ,17. We aWe compared prehospital characteristics and outcomes of subjects with descriptive characteristics across three treatment groups: 1) no IV catheter placed and no fluid administered; 2) IV catheter placed but no fluid administered; and 3) IV catheter placed and fluid administered. We report continuous variables as mean (standard deviation (SD)) or median (interquartile range (IQR)), as appropriate; categorical variables are reported as frequencies or percentages. To illustrate current practice, we plotted the predicted volume of intravenous fluid administration  across initial prehospital SBP using crude linear regression models.a priori sensitivity analyses in restricted groups’ 3) simulation of unmeasured confounders; and 4) falsification analyses to test for spurious results.Our analysis had four steps: 1) primary regression modelling of prehospital catheter and fluid on mortality; 2) a priori-determined confounders guided by past literature and theory [2, GCS, SBP), EMS diagnostic category, EMS severity index, mode of transport to receiving hospital, prehospital time intervals  and additional EMS procedures . We used a broad spectrum of measured confounders, including clustering variables, to account for treatment selection . We used independent imputed datasets for all models and combined estimates using Rubin’s rules [margins command to determine the predictive margins for each treatment group at observed covariate values [In the primary analysis to determine if prehospital catheter and fluid was associated with a lower risk of mortality (yes/no), we built a series of logistic regression models with robust (Huber-White) standard errors for regression coefficients, including unadjusted, adjusted for selected variables and adjusted for all covariates. We used d theory ,17, incl’s rules . We used’s rules . We thene values .We performed several sensitivity analyses to test the robustness of our results. First, we restricted to patients in whom ALS participated in care. This analysis limited confounding by severity . Second,We used pre-specified falsification analyses to strengthen the validity of observational research and uncover spurious associations . Our appP ≤0.05.All analyses were performed with STATA 11.0 . All tests of significance used a two-sided A total of 45,394 eligible EMS encounters were transported to a hospital during the study period Figure , of whomIn unadjusted regression models Table , we founAmong subjects with prehospital hypotension (N =554), prehospital fluid and catheter  was also associated with lower odds of hospital mortality Figure , we obseFinally, we performed multiple pre-specified falsification analyses Table . These ra priori falsification endpoints.In this population-based, observational cohort study of prehospital medical patients admitted with severe sepsis, we found that prehospital fluid administration is associated with a reduced odds of hospital mortality compared to no prehospital fluid, after multivariable adjustment. Similar to prior studies , the plaExtending prior work in the ED ,4, we foIn addition, prehospital patients with severe sepsis who received intravenous access alone were also found to have lower odds of hospital mortality. This treatment effect may result from faster triage and initiation of care in the ED. Others report that arrival by EMS reduces time to antibiotics and fluids in the ED compared to sepsis patients who arrive by non-EMS . A similThese data have important methodological and research implications. First, large treatment effects uncovered in observational studies may not always be confirmed in subsequent randomized trials . DespiteFor clinicians, the findings do not mandate that EMS begin aggressively resuscitating septic patients. Rather, the data suggest that the prehospital phase of care may be the target of future intervention trials in sepsis; such trials would be improved if there were consistent operational criteria to identifying sepsis cases. Given the variable knowledge and awareness of sepsis among emergency care providers , cliniciOut findings should be interpreted in the context of several potential limitations. First, our study did not randomly assign patients to specific prehospital resuscitation interventions. Thus, our results may be biased from a variety of measured and unmeasured confounders, such as the clinical acumen or skill of EMS with sepsis care. We determined through bias analysis that unmeasured confounders must be highly different between groups and strongly associated with hospital mortality for our findings to be rejected. We did not use propensity scores as they suffer the same limitations from unmeasured confounding, while instrumental variable analysis was considered but rejected due to the absence of a suitable instrument. We also acknowledge that different discharge practices across hospitals could bias the primary outcome of hospital mortality  and, thuIn summary, we observed that prehospital fluid administration and intravenous catheter placement were associated with decreased odds of hospital mortality in medical patients with severe sepsis. Given the adverse consequences of time delays in sepsis, these data suggest that the prehospital phase of care may be an important opportunity for early resuscitation.Prehospital fluid administration and intravenous access were uncommon among EMS patients hospitalized with community severe sepsisMean prehospital fluid administration in severe sepsis was 500 mLPlacement of intravenous access and administration of fluid was associated with a significant reduction in the odds of hospital mortality compared to no fluid or intravenous access among severe sepsis patientsThe prehospital phase of care may be an important opportunity to test early fluid administration strategies in severe sepsis


Scientific Reports5: Article number: 11344;10.1038/srep11344 published online: 06122015; updated: 03292016The Acknowledgements section in this Article is incomplete.“We thank Ben Cooper for helpful discussions, Ritva Syrjänen for pointing out useful references on the seasonality of colonization, Marc Lipsitch for advice in parametrizing the effects of previous exposures in the model and Dan Weinberger on sharing the point estimates on the Navajo colonization study, visualized in Fig. 1. E.N. and J.C. were funded by ERC grant no. 239784 and Academy of Finland grant no. 251170. P.T. was funded by the Wellcome Trust of Great Britain (Grant No. 083735/Z/07/Z).”should read:“We thank Ben Cooper for helpful discussions, Ritva Syrjänen for pointing out useful references on the seasonality of colonization, Marc Lipsitch for advice in parametrizing the effects of previous exposures in the model and Dan Weinberger on sharing the point estimates on the Navajo colonization study, visualized in Fig. 1. E.N. and J.C. were funded by ERC grant no. 239784 and Academy of Finland grant no. 251170. P.T. was funded by the Wellcome Trust of Great Britain (Grant No. 083735/Z/07/Z). This research was supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.”


G12D;Kras LSL-TrR172Hp53; Pdx1-cre (KPC) model, one of the most widely used preclinical models for this disease. We describe its application in preclinical research, highlighting its advantages and disadvantages, its potential for predicting clinical outcomes in humans and the factors that can affect such outcomes, and, finally, future developments that could advance the discovery of new therapies for pancreatic cancer.Pancreatic ductal adenocarcinoma is the most common form of pancreatic tumour, with a very limited survival rate and currently no available disease-modifying treatments. Despite recent advances in the production of genetically engineered mouse models (GEMMs), the development of new therapies for pancreatic cancer is still hampered by a lack of reliable and predictive preclinical animal models for this disease. Preclinical models are vitally important for assessing therapies in the first stages of the drug development pipeline, prior to their transition to the clinical arena. GEMMs carry mutations in genes that are associated with specific human diseases and they can thus accurately mimic the genetic, phenotypic and physiological aspects of human pathologies. Here, we discuss different GEMMs of human pancreatic cancer, with a focus on the Lox-Stop-Lox (LSL)- Drug Discovery Collection: This Review discusses GEMMs of human pancreatic cancer, particularly focusing on the KPC model, its use in preclinical research, advantages and disadvantages, and its potential for predicting clinical outcomes. The most common of these is pancreatic ductal adenocarcinoma (PDAC), an exocrine malignancy that accounts for >90% of all cases of pancreatic cancer . PDAC isKRAS oncogene (see CDKN2A) locus, point mutations in tumour protein p53 (TP53) and mutations or deletions of SMAD (Sma/mothers against decapentaplegic) family member 4 (SMAD4) are commonly found in PDAC tumours (see in situ and is the last grade before invasive carcinoma). These neoplasia grades are also well replicated in animal models .The p53 is a tumour suppressor protein that stops cells from dividing too fast, or causes damaged or mutated cells that might otherwise become tumour cells to undergo apoptosis (cell suicide). It is often mutated in pancreatic tumours, meaning that mutated cells do not undergo apoptosis and that unregulated cell division occurs instead.SMAD4 deletions in pancreatic tumorigenesis can cause cells to proliferate in an uncontrolled manner.The SMAD4 protein is a transcription factor (it regulates transcription of other genes) activated by signalling from the membrane-bound TGFβ (transforming growth factor beta) protein. The TGFβ pathway carries signals from the extracellular environment to the nucleus and affects how cells respond to such signals by inducing the production of new proteins. BRCA2 gene encodes the breast cancer type 2 susceptibility protein (BRCA2). It is essential for the repair of double-stranded DNA breaks by homologous recombination, and its loss results in chromosomal instability.The Adenosquamous: refers to a cancer type containing both gland-like cells and squamous cells .Anaplastic: cells with abnormal morphology and loss of ordered orientation, compared to normal differentiated cells.Cachexia: a wasting syndrome characterized by loss of weight, fat and muscle mass that is not reversed nutritionally.CD3: a cell surface molecule that associates with the T-cell receptor to allow its activation. The presence of CD3 on the surface of all T cells at all stages of development makes it a good marker for T cells in tissue sections.Colloid: glue like – refers to highly mucinous tumours (mucins are glycoproteins found in secretions).CRISPR/Cas: a system used for gene editing and gene regulation. The Cas9 protein causes DNA breaks, and CRISPRs  are stretches of DNA with a spacer that target a specific gene. Delivery of CRISPR guide RNAs and Cas9 into a cell allows the genome to be cut at the desired location.Dysplasia: the enlargement of a tissue or organ by the proliferation of abnormal cells.FRT recombinase system:Flp- a site-specific recombination system used to control the spatial expression of transgenes. It consists of flippase (Flp) recombinase, which targets FLP recombinase target (FRT) sequences that are placed at either end of a gene or region of interest.Fluorouracil: an antimetabolite chemotherapeutic agent that is a pyrimidine analogue that irreversibly inhibits the nucleotide synthetic enzyme thymidylate synthase.Gemcitabine: a deoxycytidine analogue used as the standard of care for pancreatic cancer. It is activated by phosphorylation, and the di- and tri-phosphate forms are responsible for its cytotoxicity. It inhibits ribonucleotide reductase, and also competes with deoxycytidine 5-triphosphate (dCTP) for incorporation into DNA during replication. It causes DNA damage and induces apoptosis.Haemorrhagic ascites: the accumulation of blood-stained fluid in the peritoneum.Irinotecan: a cytotoxic chemotherapeutic that inhibits topoisomerase 1, resulting in inhibition of DNA replication and transcription.Laparotomy: a surgical incision in the abdominal wall to gain access into the abdominal cavity.Nanoparticle albumin-bound paclitaxel (nab-paclitaxel): a paclitaxel formulation that uses albumin, the main protein of human blood plasma, to bind paclitaxel and to facilitate its transport out of the bloodstream and into the tumour. Studies show that this formulation increases the therapeutic efficacy of paclitaxel compared to its conventional formulation.Nuclear atypia: abnormal cell nuclei, e.g. often by size, shape or staining pattern.Oxaliplatin: a platinum-based chemotherapeutic agent. It leads to cross-linking of DNA, thereby inhibiting DNA synthesis and transcription.Sarcomatoid: a histological tumour subtype characterized by spindle-shaped tumour cells.Transposon (or transposable element): a short DNA sequence that can alter its position within the genome, thereby causing genetic changes.Xenograft: model generated by the injection or implantation of human cancer cells or tissues into ectopic or orthotopic sites to generate tumours in immunocompromised mice.In humans, the most frequent genetic alteration that underlies PDAC is an activating mutation of the gene see , which ogene see . In addiours see   do not correlate well with efficacy testing in humans. In fact, only a small percentage of cancer patients in Phase 1 clinical trials respond to therapies as predicted  offer an alternative to s humans . GEMMs es humans . They arG12D;Kras LSL-R172HTrp53;Pdx1-cre (KPC) model, and its use in the preclinical testing of anti-cancer agents. Finally, we discuss the importance of GEMMs in translating research findings to the clinic, and highlight their limitations and potential opportunities for their improvement.In this Review, we describe different GEMMs of pancreatic cancer and their utility for understanding the progression of PDAC and for identifying therapeutic targets. We focus in particular on one of the most widely used GEMMs of pancreatic cancer, the LSL-Kras , the deletion of Trp53 2 months after the expression of oncogenic Kras in the pancreas induces rapid multifocal tumour development  deletion in mutant-Kras-expressing pancreata blocks PanIN progression  in mice led to tumour growth in conjunction with oncogenic Kras, phenocopying the effect of genetic deletion of Lkb1 , rather than by the widely used transgenic or knock-in Cre alleles mentioned above.Another recently published model has made use of the alternate Flp-stem see . Thus fapulation . Indeed,elopment . The Flpgression . An altegression  to mutatargeting . This ap of Lkb1 . In thisKras; Pten mouse model, in which tumours have high levels of mTOR  signalling, to test the efficacy of an mTOR inhibitor , both driven by a pancreas-specific Cre, the Pdx1-cre transgene, which is expressed in all cells of the pancreas from an early stage of embryonic development , can also be used to drive tumour development in the pancreas.Traditionally, KPC mice are generated by the concomitant expression of oncogenic elopment A. KPC mielopment . These melopment B-G with 2D alone . The KPC2D alone , and met2D alone . Histopa2D alone   model, in which oncogenic Kras alone is expressed in the pancreas, as well as in KPC mice. In this setting, mice with early-onset pancreatic disease  are treated very early and prior to the onset of final-stage PDAC. In published studies, the age at enrolment varies from weaning to 10 weeks in KC mice and from weaning to 6 weeks in the KPC model, and the effect of a treatment is assessed either at pre-determined time points  or at the disease end point   . For exaurvival) . A similurvival) . The favurvival) . Nicotinurvival) . Intermiurvival) . Conversurvival) .Early intervention studies are carried out on mice of a fixed age, without predetermining whether or not they have advanced tumours. Mice are generally enrolled on a study between 10-12 weeks of age . At thisBrca1 mice at the same time point with the DNA demethylating drug 5-aza-2′-deoxycytidine (decitabine) significantly inhibited tumour growth . PEGPH20 initially proved problematic clinically, with a high rate of blood clots and other thromboembolic events observed in the PEGPH20 arm. Following a protocol amendment, the interim data from the trial was recently revealed to be promising, with increased median progression-free survival and overall response rate in the PEGPH20 arm compared with the gemcitabine/nab-paclitaxel arm. There was also a trend towards improvement in median overall survival .Targeting the stromal component, either by depleting the extracellular matrix component glycosaminoglycan hyaluronic acid (HA) or by inhibiting the matrix protein CTGF (connective tissue growth factor), was also effective in combination with gemcitabine. Whereas HA depletion by PEGPH20 resulted in improved vasculature and increased gemcitabine delivery, blocking CTGF decreased the expression of the pro-survival protein XIAP (X-linked inhibitor of apoptosis) and induced the killing of tumour cells . The depIn another preclinical study, the efficacy of nab-paclitaxel was assessed in combination with gemcitabine in a limited-duration experiment . In this+ T-cells  inhibitor, in combination with the inhibitory checkpoint antagonist anti-PD-L1 (anti-programmed death 1 ligand 1), results in the loss of p53-positive tumour cells and in the accumulation of CD3ells see  in the tells see . In anotells see . Furtheresponses . These sEstablished tumours in the KPC model undergo rapid growth. With tumours of 5-10 mm diameter, the median survival of untreated mice is around 9-11 days. As a consequence, treatment regimens tend to be of a limited duration, even where combination treatments induce a statistically significant increase in survival. Short-term studies can also be carried out with fixed durations of treatment. These enable in-depth mechanistic analyses of the therapeutic effects of a given treatment, but might also be useful in cases where longer-term treatment is not feasible. Nab-paclitaxel, for instance, is formulated using human albumin and induces anaphylaxis in mice, thereby necessitating short-term treatment . In geneIrrespective of tumour size, interventional approaches are generally labour- and resource-intensive, requiring a large mouse colony and a substantial investment of time to screen and monitor treated animals. Particularly in the case of large tumours, mice are also lost to ill health prior to enrolment, thereby extending the enrolment period, and in fixed-duration experiments, mice that do not reach the required time point owing to short survival times need to be replaced.When assessing responses to therapeutic agents, varying results can be obtained depending on tumour size. There is a lack of studies examining the differences in tumour response based on initial tumour size. However, our preliminary observations suggest that, in KPC mice with tumours of 6-9 mm diameter at the time of enrolment in the study, the tumour growth in the first 7 days post-enrolment correlates with survival, perhaps indicating that 7 days is a useful time point to assess early responses to treatment. As another example, there is generally not a significant difference in survival between vehicle- and gemcitabine-treated cohorts in KPC mice with large tumours . HoweverDuration of treatment can also affect therapeutic outcome. As previously described, Hh pathway inhibition was first reported to decrease stromal content in KPC mice with large tumours, thereby increasing gemcitabine delivery to tumours and extending survival when administered in combination with gemcitabine . UnfortuThe genetic background and the specific genetic alterations of the mice that are used in a study are two other factors to consider when evaluating response to therapy. As has been discussed earlier, the outcome of genetic and therapeutic studies can vary depending on the underlying genetic alterations in the mouse models. Understanding these differences, and identifying cohorts that are likely to respond to a given therapy, might inform the selection of patient populations in clinical trials.Improving success rates in clinical trials depends on the use of robust and predictive preclinical models. Owing to its genetic and histopathological similarity to human PDAC, the KPC model is relevant for evaluating therapies and for understanding treatment mechanisms. However, the examples mentioned above illustrate the importance of determining the best way of using preclinical models, so that the obtained results accurately reflect clinical outcome. Results obtained from studies using the KPC model suggest that mice bearing smaller tumours might be of particular relevance for survival studies because their use allows sufficient time for adverse effects to become apparent. Care must also be taken when interpreting the results of such studies, in particular focusing on change in tumour volume and not absolute tumour size.Another important question that requires consideration is whether the tumour at the end of the treatment period is the same biological entity as the initial tumour at the start of the study. Until now, tumour comparisons have been static and carried out between treatment cohorts (e.g. vehicle versus drug) because it has not been possible to obtain pre- and post-treatment tumour samples. Recently, however, a laparotomy see  method hA key strength of preclinical models is the ability to gain mechanistic insight into the tested therapies, in a manner that would not be possible in a clinical setting. For example, fixed-time-point pharmacodynamic studies can be conducted, allowing the immediate (24-48 h), intermediate (7 days) and long-term effects of treatment to be compared, for example on signalling pathways and tumour characteristics such as proliferation, apoptosis, etc. Therapies that target metastasis can be tested in early and advanced disease, and the effect of drugs on organs other than the pancreas can be assessed. Haematological and biochemical analyses can complement molecular investigations both in pharmacodynamic and survival studies. Routine imaging including high-resolution ultrasound as discussed above, but also magnetic resonance imaging (MRI), positron emission tomography and micro-computed tomography, can be carried out to evaluate tumour progression and dissemination. Preclinical testing in GEMMs also has the potential to identify tumour biomarkers that can be used to either predict drug response or to stratify patients for treatment .In addition to targeting the primary tumour and disseminated disease, studies can be conducted on symptoms, such as cachexia, which are associated with PDAC. The importance of the stroma and immune compartments in tumours can also be investigated. Recent work has, in fact, shown that the stromal compartment in PDAC might have a role in suppressing pancreatic tumours ; howevercre or the Nestin promoter (Pdx1-cre, are sometimes expressed in other tissues, resulting in off-target pathologies, such as papillomas and lymphoma. Tumour development in GEMMs can also take a long time and occurs with variable latency. Unlike xenografts, tumour monitoring might require advanced imaging, including high-resolution ultrasound and MRI. Although useful information can be obtained from these imaging modalities, they require specialist training and equipment that is not always easily available. In all, preclinical testing in GEMMs is significantly more expensive than the testing performed in in vitro culture or xenografts. Nevertheless, their advantages, as discussed here, make them a very promising preclinical platform, and a potential means of assessing complex treatment modalities and of identifying anti-cancer drug combinations to evaluate in the clinic.Despite their advantages, GEMMs have several drawbacks, including the length of time needed to generate mutant mice carrying several genetic alterations. In conditional GEMMs, such as the KPC model, genetic alterations are often activated simultaneously in a large number of cells during development in the mouse, even though they are used as models of sporadic, non-inherited human cancers. In addition, models such as KPC mice cannot be used to study the cell-of-origin of pancreatic cancer. This has required the use of alternative promoters, such as the inducible tetracycline-inducible Elastase-promoter . Tissue-Owing to their similarity to human disease, KPC mice and other GEMMs of pancreatic cancer can be excellent tools to assess therapeutics and to understand mechanisms of drug action and resistance in tumours. They have not yet been completely validated in terms of their ability to predict the outcome of trials; however, as discussed in this Review, there is extensive evidence of their relevance in preclinical research . In orde


We investigated if the efficacy of anti-L1CAM RIT can be further improved by its combination with paclitaxel (PTX).Today’s standard treatment of advanced-stage ovarian cancer, including surgery followed by a paclitaxel-platinum-based chemotherapy, is limited in efficacy. Recently, we could show that radioimmunotherapy (RIT) with In vitro cell viability and cell cycle arrest of human ovarian cancer cells were assessed upon different treatment conditions. For therapy studies, nude mice (n = 8) were injected subcutaneously with IGROV1 human ovarian carcinoma cells and received a single dose of 6 MBq 177Lu-DOTA-chCE7 alone or in combination with 600 μg PTX (31.6 mg/kg). Tumour growth delay and survival were determined. To investigate whether PTX can influence the tumour uptake of the radioimmunoconjugates (RICs), a biodistribution study (n = 4) and SPECT/CT images were acquired 120 h post injections of 2 MBq 177Lu-DOTA-chCE7 alone or in combination with 600 μg PTX.in vitro and was effective in a synergistic manner (combination index < 1). PTX increased the RIT efficacy by arresting cells in the radiosensitive G2/M phase of the cell cycle 24 h post treatment start. In vivo combination therapy including 177Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival (55 days vs. 18 days/PTX and 29 days/RIT), without weight loss and/or signs of toxicity. Biodistribution studies revealed no significant difference in tumour uptakes of 177Lu-DOTA-chCE7 72 h post injection regardless of an additional PTX administration.Lu-DOTA-chCE7 in combination with PTX revealed a significantly decreased cell viability of ovarian carcinoma cells 177Lu-RIT with PTX is a more effective therapy resulting in a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications.Combination of anti-L1CAM The online version of this article (doi:10.1186/s13550-014-0054-2) contains supplementary material, which is available to authorized users. With an estimate of 21,980 new cases in the US alone, ovarian carcinoma (OC) represents the fifth most common cause of cancer deaths in female population in 2014 . Today’sBesides conventional chemotherapy, numerous monoclonal antibodies (mAbs) have been developed for targeted therapies for the future management of ovarian cancer . Althoug90Y-labelled radioimmunconjugate (90Y-muHMFG1) for the treatment of OC has advanced to a clinical phase III trial. Unfortunately, no improvement in extending survival or time to relapse could be achieved in particular due to a missing dosimetric approach. Thereby, retrospective analysis revealed that tumour absorbed doses had been too low [In order to improve the efficacy of mAb-based therapies, radioimmunotherapy (RIT) is considered to be an attractive strategy for the treatment of OC . While R too low ,14.90Y and the application of a non-internalising antibody (anti-MUC1) are considered further reasons for the limited clinical outcome.The use of the high-energy-emitting radionuclide The L1 cell adhesion molecule (L1CAM) was originally described as a protein of the nervous system and is highly expressed on numerous tumours such as neuroblastoma , colon cKD ≈ 10−10 mol/l) near an RGD sequence in the sixth IgG-like domain of L1CAM, inhibiting tumour cell growth in vitro and in vivo [177Lu-labelled variant of mAb chCE7 showed high efficacy in a xenograft model of disseminated ovarian carcinoma [chCE7 is a chimeric monoclonal antibody that is directed against the L1CAM cell surface antigen. mAb chCE7 binds with high affinity  can be advantageous compared to monotherapies -29. PTX 177Lu-RIT against ovarian carcinoma can be further increased by its combination with the radiosensitising taxane PTX.In this study, we investigated whether the efficacy of previously developed anti-L1CAM 2 in RPMI 1640 medium at 37°C. The medium was supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml fungizone . mAb chCE7 is a IgG1-subtype chimeric monoclonal antibody (human κ light chain and human γ1 heavy chain). It was produced in HEK293 cells and purified from cell culture supernatant using a protein G-Sepharose column  as described by Grünberg et al. [IGROV1 human ovarian cancer cells were kindly provided by Dr. Cristina Müller  and analysed by STR profiling . IGROV1 cells were maintained in a humidified atmosphere containing 5% COg et al. . An unsp3PO4 solution and was incubated for 16 h at 4°C. Excess ligands were removed and buffer was exchanged into 0.25 M CH3COONH4 (pH 5.5) using a NAP-5 column . Immunoconjugates were stored at −80°C.Ligand substitution was performed as previously described by Fischer et al. . For lig177Lu  was utilised for radiolabelling 1 to 3 days post calibration date. Briefly, a reaction mixture containing 250 to 900 μg of the immunoconjugates and 200 to 600 MBq 177Lu was incubated in 0.25 M CH3COONH4 buffer (pH 5.5) for 1 h at 37°C.The average number of coupled chelators per mAb was determined by mass spectrometry as previously described . 177Lu . The flow rate of the mobile phase  was set to 1 ml/min  were purified via FPLC size exclusion chromatography on a Superose 12 column  in phosphate-buffered saline (PBS) with a flow rate of 0.5 ml/min. Both radiolabelled chCE7 and unspecific control IgG eluted at a retention time of 21 min. In order to test the stability of 5/well) and incubated for 24 h. The medium was removed and cells were incubated with the accordant ½ half-maximal inhibitory concentration  or IC50 (10 nM) of PTX for 24 h at 37°C. PTX ½ IC50 was calculated based on the experimentally determined IC50 value using a 3--2,5-diphenyltetrazolium bromide (MTT) cell viability assay , IGROV1 cells were seeded in a 96-well plate and incubated for 24 h at 37°C. After adhesion, cells receiving combination treatment were incubated with the accordant ½ IC50 (5 nM) or IC50 (10 nM) PTX concentrations for 24 h at 37°C in order to maximise the amount of cells being arrested in the G2/M phase of the cell cycle. Cells were then washed with PBS and treated with 100 μl (0.02 to 42 MBq/ml) 177Lu-DOTA-chCE7 for 4 h on ice. Subsequently, cells were washed and incubated in culture medium at 37°C. Cell viability was determined when 20 μl of filtered MTT solution  was added to each well followed by incubation for 2 h protected from light. The medium was removed and the formed formazan crystals were dissolved in 200 μl dimethyl sulfoxide (DMSO). The absorbance (OD) was determined at a wavelength of 560 nm in a microplate reader . Results are expressed as percentage of viable cells compared to the control.In order to determine the 6 IGROV1 cells  into the right flank. Eight days post tumour cell inoculation, therapy experiments started (mean tumour volume = 60 ± 30 mm3). Mice were injected with a) 6 MBq (50% maximum tolerated activity (MTA), 25 μg, 100 μl) 177Lu-DOTA-chCE7, b) 6 MBq 177Lu-DOTA-control IgG  or c) PBS into the tail vein. MTA of 177Lu-DOTA-chCE7 was determined elsewhere [177Lu-DOTA-chCE7 is reached at 48 to 72 h after injection of RIC (i.v.), PTX was administered 24 h post RIT in order to synchronise the maximum tumour uptake of the radiolabeled mAb and PTX-induced cell cycle arrest in the G2/M phase. Concentration was chosen based on previous experiments determining 600 μg/mouse as a reliable dosage for PTX administration in combination with RIT [All animal experiments were approved by the cantonal committee on animal experiments and permitted by the responsible cantonal authorities (permission numbers 75528 and 75535). The studies were conducted in compliance with the Swiss laws on animal protection. For survival studies, groups of eight female CD1 nude mice  were injected subcutaneously (s.c.) with 7 × 10lsewhere . Twenty-with RIT ,35.V = (A × B2)/2, where A is the smaller diameter and B the wider diameter of the tumour. The relative tumour volume (RTV) was calculated with Vx/V0 . Relative body weight (RBW) was measured with Wx/W0 . Animals were euthanised if the tumour volume exceeded 1,000 mm3 or observed weight loss was greater than 20%. In order to avoid bias in the test results, treatment information was blinded to the tester during the course of the therapy.Animals were examined two to three times a week and weighed, and tumour volumes were measured with 6 IGROV1 cells at the age of 5 weeks. Fourteen days post tumour cell inoculation, 0.85 MBq  177Lu-DOTA-chCE7 was injected into the tail vein. Twenty-four hours later, mice received either 600 μg PTX  i.p. or 300 μl PBS i.p. Control mice were injected with 0.85 MBq 177Lu-DOTA-control IgG . Mice were sacrificed 72 h post RIC administration, and organs as well as tumours were weighed and counted for radioactivity in a gamma counter . Results are expressed as percentage of the injected activity per gram of tissue weight (%IA/g).For biodistribution studies, groups of four female CD1 nude mice  were injected s.c. with 7 × 10177Lu-DOTA-chCE7  was injected into the tail vein of tumour-bearing nude mice 24 h prior to i.p. administration of 600 μg (31.6 mg/kg) PTX or PBS. Control mice received 177Lu-DOTA-control IgG. SPECT/CT scans were performed 96 h (177Lu-DOTA-control IgG) and 120 h  after RIC administration. SPECT data were reconstructed by HighSPECT software . Reconstruction of CT data, fusion with SPECT data and analysis were performed by InVivoScope postprocessing software .SPECT/CT imaging studies were performed in a NanoSPECT/CT system . p < 0.0083. Student’s t test  was used for comparison of tumour volumes and biodistribution experiments. Statistical significance was defined as p < 0.05. In vitro data was analysed via combination index calculations  + ). Thereby, concentrations required to produce a given effect are determined for drug A  and drug B . C,xA and CxB, are the concentrations of A and B contained in combination that provide the same effect. Synergy is determined for CI < 1, additivity for CI = 1 and antagonism for CI > 1 [Statistical analysis of the survival experiment was performed via a log-rank test. Bonferroni correction was used to determine statistical significance for multiple comparisons. Significance was defined as r CI > 1 .177Lu labelling was 240 to 600 MBq/mg protein. Immunoreactivity was proven by the Lindmo method (50% to 87%).Mass spectroscopic analysis of the DOTA-to-mAb ratios for mAb chCE7 and the control IgG revealed that an average of 2.7 to 3.1 (chCE7) or 3.1 (control IgG) chelators were coupled to an intact antibody molecule. Specific activity obtained upon 50 (5 nM) or IC50 (10 nM) PTX concentrations for 24 h. An increased amount of IGROV1 cells arrested in the G2/M phase of the cell cycle could be observed for cells treated with 10 nM PTX  and ½ IC50 (5 nM) of PTX as a basis for combination treatments. One-half IC50 was calculated based on the experimentally determined IC50  and 2.9 ± 1.2 MBq/ml (177Lu-DOTA-chCE7 + 10 nM PTX). In order to determine synergistic or additive effects, combination index (CI) calculations as published by Zhao et al. were used [50) increases the cytotoxic effect of 177Lu-DOTA-chCE7 in a synergistic manner (CI < 1). In order to provide additional information about the long-term colony-forming ability of IGROV1 cells after mono- (177Lu-DOTA-chCE7) or combined (PTX + 177Lu-DOTA-chCE7) treatments, colony assays were assessed. Results indicate that the cytotoxicity of 177Lu-DOTA-chCE7 can be increased by the additional application of paclitaxel (½ IC50) 24 h prior to RIT  to 0.75 MBq/ml for combined treatment (PTX + 177Lu-DOTA-chCE7). Simultaneous administration or application of PTX 16 h after 177Lu-DOTA-chCE7 did not increase the cytotoxicity effects of RIT necessary for 50% cell growth inhibition.In order to investigate whether PTX can sensitise IGROV1 cells towards subsequent l Figure . Combineere used . For a c177Lu-DOTA-chCE7 and PTX results in delayed tumour growth and prolonged survival compared to monotreatments, a therapy experiment was performed in nude mice. Xenografts were generated by s.c. injection of IGROV1 human ovarian carcinoma cells into female CD1 nude mice.To investigate whether a combined treatment of 177Lu-DOTA-chCE7 (25 μg) followed by 600 μg PTX 24 h later. Control mice received either 6 MBq (25 μg) of a 177Lu-labelled unspecific control IgG or PBS. Mice were monitored two to three times a week with end point criteria set as weight loss >20% or increased tumour volume >1,000 mm3. Average RTVs, average RBWs and Kaplan-Meier survival plots were recorded. Tumour growth curves were stopped at the exclusion day of the first mouse in each group. Untreated control mice showed a fast increase in tumour volume from the beginning of the therapy experiment, indicating an uninhibited tumour growth  demonstrated the most pronounced delay in tumour growth. The mean RTV < 1 between days 4 and 18 post treatment start indicates tumour shrinkage. Complete tumour clearance or shrinkage below detection limits was observed in seven out of eight mice until day 13. During this time, a reduced tumour burden with a significant decrease in average RTV on day 13 was observable for mice treated with anti-L1CAM combination therapy compared to all other treatment groups . Additionally, no weight loss >20% or signs of distress could be observed at any time during the therapy experiment. Combination of 177Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival compared to mice that received only 177Lu-RIT or PTX  up to 55 days for the combination therapy. Additionally, a significantly prolonged survival could be shown for mice receiving anti-L1CAM combination therapy compared to untreated controls (PBS) and mice that received unspecific treatments . No significant difference in overall survival could be observed between PTX monotreatment and the untreated control (p = 0.29).Eight days post implantation of tumour cells, mice received an i.v. injection of 6 MBq 177Lu-RIT alone increased overall survival significantly compared to mice that received only PBS (p = 0.0017). A regular increase in mean RBW for mice that received the combination of 177Lu-DOTA-chCE7 and PTX could be observed until day 37 post treatment start indicating no therapy-induced weight loss and/or signs of toxicity  were injected i.v. with 0.85 MBq 177Lu-DOTA-chCE7 14 days post s.c. implantation of IGROV1 tumour cells. Twenty-four hours post 177Lu-DOTA-chCE7 administration, one group of mice additionally received 600 μg PTX (i.p.). Control mice (n = 5) were injected with a non-binding 177Lu-DOTA-control IgG with or without PTX treatment. Accumulated radioactivity in tumour tissue was high after 72 h (49.6% ± 11.9%) for mice injected with 177Lu-DOTA-chCE7 compared to reasonably low uptakes for all non-targeted organs and the blood pool , implying that PTX has no effect on the tumour uptake of 177Lu-DOTA-chCE7. Similarly, PTX had no influence on the accumulation of 177Lu-DOTA-chCE7 in all other organs and the blood pool. As expected, the tumour uptake of the 177Lu-DOTA-control IgG was low regardless of a following PTX administration. For 177Lu-DOTA-control IgG, we observed a longer retention time in the blood pool compared to 177Lu-DOTA-chCE7. Very low radioactivity in the bones indicates maintained stability of the 177Lu-DOTA complex after 72 h post injection. We further analysed the distribution of 177Lu-DOTA-chCE7 in subcutaneous IGROV1 tumour-bearing nude mice at 120 h post RIC administration via SPECT/CT. Injection of 177Lu-DOTA-chCE7  were combined in a synergistic manner.These results agree with previous observations that low PTX concentrations (<10 nM) induced an increased amount of apoptosis without evidence for existent G2/M arrest compared to a control. This effect might be caused by the fact that lower PTX concentrations (<10 nM) do not completely saturate PTX binding sites in a part of the cell population, which in turn leads to progression of the cell cycle, chromosomal instability and induction of apoptosis . For inty90Y-labelled mAb B3 and PTX combination therapy resulted in a significantly prolonged survival of human epidermoid carcinoma-bearing mice. Milenic et al. [213Bi-trastuzumab) was combined with PTX in a human colon carcinoma tumour model. However, the radioisotope 177Lu is a more suitable candidate for RIT against smaller tumours that frequently appear in ovarian cancer. 177Lu shows an increased half-life of 6.7 days compared to 213Bi (45 min), thereby matching the slow pharmacokinetics of IgGs when injected intravenously. Furthermore, an intravenous application of the RIC might have advantages in terms of targeting distant metastasis beyond the peritoneal cavity. Thereby, 177Lu lower tissue penetration range (≈2 mm) is likely to be superior to 90Y in the treatment of small disseminated ovarian cancer tumour nodules [177Lu-DOTA-chCE7 led to an increased therapeutic efficacy in a xenograft model, resulting in a significantly prolonged overall survival. Thereby, PTX was administered 24 h post RIT in order to adapt peak concentrations of 177Lu-DOTA-chCE7 and PTX in the tumour. Results indicate that PTX has the ability to increase the cytotoxic effects on IGROV1 tumour cells induced by 177Lu-DOTA-chCE7. Mice that received only PTX monotreatment showed no significant difference in tumour growth delay and overall survival compared to untreated control mice, implying that only subcytotoxic PTX concentrations  were used. Observation of insignificant tumour suppression of PTX monotreatments compared to untreated controls is in line with previous studies demonstrating the limited subtherapeutic effect of low-dose PTX treatments [Increased therapeutic efficacy of RIT upon combination with PTX has been previously shown by Jang et al.  when antc et al.  demonstr nodules . Our anieatments .177Lu-DOTA-chCE7 uptake in either way. As expected, tumour uptake of the control RIC with or without PTX application was very low, indicating only a non-specific accumulation. Low non-specific tumour uptakes were consequently accompanied by higher remaining levels of activity in the blood pool.Biodistribution studies demonstrated high tumour uptakes for the specific RIC correlating with low remaining levels of activity in the blood pool 72 h post RIC injection, regardless of an additional PTX administration. Thus, PTX did not influence the 3) showed decreased interstitial fluid pressure and increased blood vessel permeability resulting in higher RIC accumulation in the tumour. In our studies, increased tumour accumulation was not observed, supporting our assumption that cell cycle arrest in the G2/M phase played a major role during in vivo combination therapy. However, additional effects on the tumour microenvironment caused by PTX cannot be fully excluded, since cellular and tumour microenvironmental effects are known to complement each other [Jang et al.  demonstrch other .177Lu-DOTA-chCE7 was increased by the introduction of PTX into the treatment scheme, no weight loss or a decreasing number of white blood cells was induced (data not shown).Even though the therapeutic efficacy of While the application of a third chemotherapeutic is thought to result in unjustifiable toxicities for patients, RIT has shown to be well tolerated with low toxicity levels. Therefore, RIT/paclitaxel/platinum-based chemotherapy combination offers an alternative treatment strategy that may improve the efficacy of a first-line platinum-paclitaxel treatment. Nevertheless, such an alternative treatment strategy has to be verified in large, randomised clinical trials. Since patterns of platinum resistance and mechanisms of action for microtubule-stabilising agents do not necessarily interact, patients with platinum-refractory, platinum-resistant disease or platinum-resistant relapse might benefit from a RIT/paclitaxel combination. Again, this has to be verified in clinical trials.Malignant ascites are frequently occurring in patients with advanced ovarian cancer with only limited treatment options. So far, catumaxomab, a trifunctional mAb, is used for the effective treatment of EpCAM-positive tumour cells in the peritoneal cavity . An effe177Lu-DOTA-chCE7 RIT may provide a potential clinical setup against residual ovarian tumour nodules after first-line tumour resection. Patients may therefore benefit from decreased side effects during therapy and possible increased therapeutic efficacy.It is well known that patients suffer from severe side effects (e.g. neuropathy and myelosuppression) induced by high concentrations of PTX during treatment cycles . Since R


Significant inhibitory impact of metformin was observed in MCF-7, HeLa, and SKOV-3 cells, suggesting an antiproliferative property of metformin against breast, cervical, and ovarian tumour cells, respectively, with the breast tumour cells, MCF-7, being the most responsive. In vivo assessment was subsequently carried out, where mice with breast tumours were treated with metformin (20 mg/kg body weight) or sterile PBS solution for 15 consecutive days. No inhibition of breast tumour progression was detected. However, tumour necrosis was significantly increased in the metformin-treated group, accompanied by decreased capillary formation within the tumours. Thus, despite the lack of short-term benefit of metformin against tumour progression, a preventive role of metformin against breast cancer was implicated, which is at partially attributable to the attenuation of tumour angiogenesis.Metformin is one of the most widely prescribed antidiabetics for type 2 diabetes. A critical role of metformin against tumorigenesis has recently been implicated, although several studies also reported the lack of anticancer property of the antidiabetics. Given the controversies regarding the potential role of metformin against tumour progression, the effect of metformin against breast, cervical, and ovarian tumour cell lines was examined followed by  In recent years, epidemiological analyses have indicated a positive association between long-term diabetes and elevated risk of malignant neoplasms . In part in vivo assessment of metformin on tumour growth.Metformin is one of the most efficacious and safe front-line antidiabetics for type 2 diabetes (T2D). In addition to its antiglycaemic impact, recent reports also implicated critical role of metformin in tumourigenesis , 2. Indeμg/mL streptomycin . Cells were seeded at a density of ~5000 cells per well in 96-well plates and maintained at 37°C under standard culturing conditions. Cells were exposed to a series of concentrations of metformin  continuously and cell viability was determined at the end of 24 h and 5 days using a cell counting kit-8 .Human breast (MCF-7), ovarian (SKOV-3), and cervical (HeLa) cancer cells were cultured in DMEM media  supplemented with 10% foetal bovine serum , 100 units/mL penicillin, and 100 3, mice were randomly assigned to either control group or metformin-treated group. Local injection of metformin (20 mg/kg body weight) or sterile PBS was administered for 15 consecutive days. Changes of body weight were monitored and tumour volumes were measured and corrected according to standard formula [Xenograft breast tumour models were established by injecting MCF-7 cells into 6-week-old female BALB/c nude mice . Once the tumour size reached ~100–150 mmμm) were cut onto microscope slides. Haematoxylin and eosin (H&E) staining was employed to examine tumour morphology and immunofluorescent staining using an antibody raised against von Willebrand factor  was also carried out to evaluate capillary formation. The staining data were analysed with a fluorescent microscope  and fluorescent intensity was quantified using ImageJ software .15 days after initial injection, tumours were dissected and fixed in 4% paraformaldehyde before being paraffin embedded. Consecutive sections  or metformin  was administered daily at the tumour site for two weeks. No changes of tumour volume were detectable between the control and metformin-treated groups  for 15 days. In fact, the effect of metformin against breast cancer has long been extensively investigated albeit contradictory as summarized in a recent review [ per se. However, this result may not reflect long-term effect of the drug as the necrosis area was considerably larger in tumours obtained from metformin-treated mice.Subsequent in vivo, a preventive role of metformin against breast cancer was implicated, which is at least partially attributable to the attenuation of tumour angiogenesis. Further investigation is required to evaluate whether the antiangiogenic effect of metformin is tumour-specific, particularly since metformin is widely prescribed as an antidiabetic and T2D patients have an elevated risk of vascular disorders. Furthermore, considering the diversity of metformin action, the exact mechanisms underlying the antiangiogenic property of metformin are also required to be elucidated.In addition, significant attenuation of capillary formation was also evident from the metformin-treated group, consistent with a previous report proposing an AMPK/mTOR-dependent antiangiogenic effect of metformin on ovarian cancer . Thus, d


Here we show that the VDVF vent fluid is generated by interaction of seawater with a mafic and ultramafic basement which precipitates talc on mixing with seawater. The heat flux at the VDVF is measured at 487±101 MW, comparable to the most powerful magma-driven hydrothermal systems known, and may represent a significant mode of off-axis oceanic crustal cooling not previously recognized or accounted for in global models.The Von Damm Vent Field (VDVF) is located on the flanks of the Mid-Cayman Spreading Centre, 13 km west of the axial rift, within a gabbro and peridotite basement. Unlike any other active vent field, hydrothermal precipitates at the VDVF comprise 85–90% by volume of the magnesium silicate mineral, talc. Hydrothermal fluids vent from a 3-m high, 1-m diameter chimney and other orifices at up to 215 °C with low metal concentrations, intermediate pH (5.8) and high concentrations (667 mmol kg The Von Damm Vent Field comprises 85–90% talc, with hydrothermal fluids of unusual chemistry at 215 °C. Here, the authors show that the mineralogy results from seawater mixing with hydrothermal fluids and the heat output may represent a mode of crustal cooling not fully accounted for in global models. Hydrothermal activity at mid-ocean ridges is dominated by basalt-hosted, high-temperature, metal-rich vent systems driven by magmatic activity24James Cook in April 2010, which hosts a community of hydrothermal vent fauna similar to those at the Mid-Atlantic RidgeA new class of hydrothermal system, the Von Damm Vent Field (VDVF), was discovered in the Caribbean during cruise JC044 of the RRS Main Cone onlaps the surrounding calcareous sediment—a relationship reversed at the base of the southern mound, indicating an increase in the age of the mounds towards the south 2) as the dominant phase. Shifts in peaks of the 001 plane from 9.604–9.627 Å for the air-dried mounts to 9.339–9.525 Å for the glycolated mounts show the presence of up to 10% of a smectite clay that is interlayered within the crystalline talc structure. The presence of peaks at 1.529 and 1.532 Å (for the 060 plane) indicate a tri-octahedral structure for the smectite clay interlayers  and MgO, (25–34 wt.%). Average base-metal concentrations in the hydrothermal chimneys are 1,463 p.p.m. Cu, 239 p.p.m. Zn and 112 p.p.m. Pb. In contrast, concentrations in samples of mound talus are an order of magnitude lower with averages of 241 p.p.m. Cu, 48 p.p.m. Zn and 15 p.p.m. Pb, consistent with the observed oxidation and loss by dissolution of sulphides in the talus samples.The high proportions of talc and microcrystalline silica are reflected in the whole-rock geochemistry  by the hN)(/SmN) ratios ranging between 0.9 and 10.4, with an average of 3.3 for chimneys and 3.1 for mound talus (N)(/YbN)(/√(SmN)( × GdN), ranges between 6 and 228, with an average of 99 for the chimneys and 58 for the mounds 87Sr/86Sr ratios of between 0.702902 and 0.703657 (Rare earth element (REE) patterns for talc, separated from hydrothermally active chimneys and mound talus samples, have shallow U-shaped profiles dominated by a large and positive europium anomaly . Enrichmnd talus . The REEe mounds . The VDV0.703657  and 3.87Sr/86Sr ratios indicate precipitation following mixing of the high-temperature VDVF vent fluid with a high proportion of seawater (of at least 10:1). Chlorinity in the end-member VDVF fluid of 667 mmol kg−1 is significantly elevated in comparison with seawater (546 mmol kg−1), enhancing the complexation of LREEs in relation to mid-RREs (MREEs) and HREEs in hydrothermal solutions at high temperatures and pressures, resulting in elevated LREEtalc/MREEtalc ratiosThe U-shaped REE patterns and positive Eu anomalies in the chimney and mound material indicate talc precipitation from VDVF vent fluid that has some similarities to high-temperature ‘black smoker' vent fluid REE chemistry87Sr/86Sr ratios for the talc further indicate that a significant component of seawater is mixed with the vent fluid during talc precipitation.Other examples of seafloor talc deposits, reported from the St Paul and Conrad fracture zones, are inferred to have precipitated from the interaction of hydrothermal fluid with either seawater or a mafic protolith17We conclude from the petrographic and geochemical evidence that the VDVF talc is a primary precipitate from a hydrothermal fluid mixed with seawater. To date, no other seafloor, talc-dominated, active hydrothermal vent field has been reported, making the discovery of the VDVF a new and unique class of hydrothermal system.−1 (ref. −1) and Li (241 μmol kg−1) are much higher than those generated by phase separation of seawater alone , indicating a process of brine concentration.To explore whether the VDVF is currently precipitating talc, we sampled and analysed the composition of the vent fluids escaping from three different chimneys with a maximum measured temperature range of 108–215 °C . It shouer alone , indicat0.703657  and 3. B derived . Variati2S concentrations at the VDVF are indicative of minimal magmatic contribution, consistent with the lower temperature of the end-member vent fluid and the ridge-flank setting of the vent field.The low metal concentrations as well as the near-neutral pH of the fluids venting at VDVF are consistent with temperatures in the water–rock reaction zone, which are significantly cooler than the ∼500 °C calculated for ‘black smoker' ventsIncreases in chlorinity for hydrothermal fluids interacting with an ultramafic basement have been suggested to occur in a number of ways: rock alteration, phase separation and brine and halite addition2262−1 (see below) for which the increase in chlorinity requires brine expulsion following serpentinization of pure forsterite at a rate of at least 470 kg s−1. Alternatively, the entrainment of an early-formed brine or halite phase is consistent with a cooling hydrothermal system, while serpentinization is compatible with the tectonic setting of the VDVF on slowly exhumed lower-crust and upper-mantle rocks. Both processes are ultimately constrained by the availability of residual brine, halite or fresh peridotite.We have calculated a mass flux of end-member hydrothermal fluid at VDVF of ∼500 kg s+ is produced at a rate greater than it is used up in hydrolysis reactions, resulting in lower pHA measured pH of 5.8 (at STP) for the VDVF end-member fluid is significantly higher than the observed range pH 2.5–3.5) for sulphide-rich, high-temperature ‘black smoker' vent fluids. It is also lower than the alkaline, low-temperature fluids vented at the ultramafic-hosted Lost City Vent Field.5 for suWith its intermediate pH, 215 °C temperature and setting on lower-crustal and upper-mantle rocks, the fluids venting at the VDVF are consistent with moderate-temperature interaction within a gabbro/peridotite basement .2O-HCl-(Al2O3)-MgO-SiO2 system. A fluid composition around the eutectic between talc and amorphous silica would be influenced by slight changes in Mg or Si activity, brought about either by precipitation of one or the other phase, or by fluctuating seawater proportions within the mound. Silica is saturated in the end-member fluid, therefore would precipitate when no seawater is available to provide Mg for talc precipitation. Such dynamic conditions, which are well documented at other sites333487Sr/86Sr ratios for the talc (0.706313–0.709168), which lie between the value of modern seawater (0.7092) and the VDVF end-member hydrothermal fluid  located ∼700 m to the east of the currently active site. The current VDVF represents the latest stage in talc precipitation and hydrothermal circulation in the mafic and ultramafic basements beneath the Mt Dent OCC over tens of thousands of years.Hydrothermal venting on ocean crust more than 1 Ma in age has only been observed previously as either low-temperature (<10 °C) diffuse flow of reduced fluids3While talc-rich deposits of postulated hydrothermal origin have been reported beforeAt both sites, botryoidal talc, with similar REE patterns to the VDVF talc, was recovered from a tectonically exhumed gabbro–peridotite basementin situ fluid temperatures and the diameters of vent orifices at the VDVF, we have calculated a focused hydrothermal heat flux of 487±101 MW, produced from the venting of ∼500 kg s−1 of end-member fluid (TAG vent field (758 MW) on the Mid-Atlantic Ridge40By measuring vertical velocities of vent fluid as it exits the seafloor, er fluid . Despite2S and low concentration of dissolved metals in the VDVF vent fluid are all features consistent with hydrothermal circulation driven by residual heat within the tectonically uplifted lower crust and upper mantleWhile hydrothermal activity is effective at extracting heat from the magmatic ridge axis43Isis and HyBIS) during RRS James Cook cruises JC44 and JC82.Sonar data, photographic and video imagery and samples of rocks, minerals, fluids and temperatures were acquired using remotely operated vehicles (2S being determined at STP (25 °C) immediately after recovery of the titanium syringes on the surface. Anions were determined using ion chromatography, and cations using inductively coupled plasma atomic emission spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) at NOCS. Data quality was assessed using the ‘Mottl Vent Fluid Database' protocolsHigh-temperature fluids were sampled using titanium gas-tight syringes at three different high-temperature chimneys . Fluid e2 blocks of untreated broken surfaces; the second was a high-resolution LEO 1450-VP SEM operating at 20 kV and imaging carbon-coated polished thin sections. Crystallographic analysis was made using X-ray diffraction with Cu Kα radiation 2θ ranging between 2° and 76°, at steps of 0.02° and at rates of 0.02° per s. Unorientated air-dried mounts and glycolated mounts were scanned in the ranges 0–20° and 0–40°. Randomly orientated mounts were prepared and scanned in the range 57–62° to identify the peak signifying the (060) crystallographic plane.Petrographic analyses were conducted using transmitted and reflected light microscopy on polished thin sections. SEM was conducted using two different systems: one using a low-resolution Hitachi TM1000 Desktop SEM operating at 15 kV, imaging 1-cm87Sr/86Sr ratios were obtained using a thermal ionization mass spectrometer. Fluid and host rock samples were analysed by running through Sr-specTm resin to obtain 1 μg of strontium. Samples of strontium (200 μg) were obtained from pure talc by running through cation resin AG50-X8 200–400 and then through Sr-specTm resin. External reproducibility of analysis was checked by analysing standard NBS987 every 11 samples and is reported here with an average of 0.710250 (1σ=0.0000053), which is within an error of the accepted value of 0.710254.X-ray fluorescence (XRF) analysis was conducted on fused beads in a 10:1 ratio of sample powder to lithium tetraborate flux. Precision was determined from repeat analysis of standards UB-N, BRR-1 and OPY-1, and gives errors of <10%. Talc mineral separates were handpicked and washed in a weak acid solution and rinsed in de-ionized water. Following hydrofluoric and nitric acid digestion, the samples were analysed for trace element and REE analyses using ICP-MS. Standards included international reference materials: BIR-1, JB1a, JGB1, JB-3 and BHVO-2. Precision was determined from repeat analysis and gives errors in the range of 0.21–5.71%. Whole-rock analysis was conducted using both ICP-MS and inductively couple plasma atomic emission spectroscopy, and calibration was made using synthetic multielement standards. Modelling was carried out using Geochemist's WorkbenchFocused hydrothermal flow rates were calculated from high-definition video footage of the vertical velocity of particles entrained within venting hydrothermal fluid.Individual particles entrained in the exiting vent fluid were filmed over a rise height of ∼1 m and their velocity calculated from frame to frame. The distance travelled by the particles was measured against two parallel laser beams separated by a constant distance of 10 cm and shone through the rising vent fluid. The fluid exit velocities at each vent orifice were determined by measuring multiple particles in the upflowing fluid across the entire dimensions of the vent orifices. Different particles entrained in the up-flow zone from each orifice displayed only slight variation in their velocities, indicating that the flow rates are relatively uniform for fluids exiting each vent orifice . The vel48495051This method models the vent orifices as circles, and makes the assumptions that the fluid is emanating from the vent at a constant velocity across its diameter, the temperature is uniform across the vent orifice and the particle velocities recorded were representative of the flow velocity. Specific heat capacity of the venting fluids was calculated using specific densities and thermal capacities for seawaterHow to cite this article: Hodgkinson, M. R. S. et al. Talc-dominated seafloor deposits reveal a new class of hydrothermal system. Nat. Commun. 6:10150 doi: 10.1038/ncomms10150 (2015).Supplementary Figures 1-2 and Supplementary Table 1


The staggered grid arrangement together with MAC method was employed to solve the governing equations. It is found that the fluid flow and the heat transfer can be controlled by the thickness of the bottom wall, the thermal conductivity ratio, and the Rayleigh number.Conjugate natural convection-conduction heat transfer in a square enclosure with a finite wall thickness is studied numerically in the present paper. The governing parameters considered are the Rayleigh number (5 × 10 The beautiful hexagonal patterns can be obtained by a simple experiment first conducted by Henri Bénard, a French physicist, in 1900. Later, in 1916, Lord Rayleigh proposed his theory of a feedback coupling resting on buoyancy: a fluid particle hotter than its environment encounters ever colder fluid as it rises, which leads to the instability. The critical Rayleigh numbers, which signal the onset of natural convection in rectangular boxes heated from below and cooled from above, have been obtained theoretically by Davis . CorcionThe literatures indicated that most of the reported works on Rayleigh-Bénard convection do not study the effect of a conductive bottom wall. Applications of conductive bottom wall can be found, for example, in high performance insulation for buildings. When the conductivities of the wall and fluid are comparable and the wall thickness is finite, conduction-convection analysis is necessary. This coupled conduction-convection problem is known as conjugate convection. Conjugate natural convection in a rectangular enclosure surrounded by walls was firstly examined by Kim and Viskanta , 5. TheiThe investigation of the effect of the conductive bottom wall on convective flows in a square enclosure has not received much attention. Recent works include those of Varol et al.  and SaleTh, and the top surface of the enclosure is cooled to a constant temperature Tc, while the vertical walls are kept adiabatic.A schematic diagram of a square enclosure with a finite wall thickness is shown in f and w stand for the fluid and the wall, respectively. No-slip condition is assumed at all of the solid-fluid interfaces. Using the following nondimensional variables:Thermophysical properties of the fluid in the flow field are assumed to be constant except the density variations causing a body force term in the momentum equation. The Boussinesq approximation is invoked for the fluid properties to relate density changes to temperature changes and to couple in this way the temperature field to the flow field. Under the above assumptions, the governing equations for steady natural convection flow using conservation of mass, momentum, and energy can be written as∂u∂x+∂v∂ykw/kf is the thermal conductivity ratio. The physical quantities of interest in this problem are the average Nusselt number defined byThe values of the nondimensional velocity are zero in the wall region and on the solid-fluid interfaces. The boundary conditions for the nondimensional temperatures are the following:i is used to represent the coordinate X and ND is the number of nodal points in the Y-axis in the wall.Staggered grid arrangement together with the Marker and Cell (MAC) method  is adoptRegular and uniform grid distribution is used for the whole enclosure. The effect of grid resolution was examined in order to select the appropriate grid density as demonstrated in D ≤ 0.4, the thermal conductivity ratio, 0.5 ≤ Kr ≤ 10, and the Rayleigh number, 5 × 103 ≤ Ra ≤ 106.The analyses in the undergoing numerical investigation are performed in the following range of the associated dimensionless groups: the wall thickness, 0.02 ≤ D for Ra = 105 and Kr = 1 on the thermal fields and flow fields in the fluid and in the bottom solid wall. As can be seen, the parameter D affects the fluid and the solid temperatures as well as the flow characteristics. The strength of the flow circulation of the fluid is much higher for a thin solid bottom wall. A circular main cell is formed at a thin bottom; then, the main cell shape becomes elliptical and finally breaks up into dual contrarotative cells at D = 0.4. This is because the fluid adjacent to the hotter wall has lower density than the fluid at the middle plane. As a result, the fluid moves upward due to the Archimedes force from the middle portion of the top wall. When the fluid reaches the upper part of the enclosure, it is cooled, so its density increases; then, the fluid flows downward at the left and the right planes of the enclosure. This creates a successive cell that is well known as Bénard cells. It is important to note that the Rayleigh number in the present study is based on the total height of the enclosure.5 and D = 0.3. Three different conductivity ratios are selected: Kr = 0.5, Kr = 1, and Kr = 10. It is observed that two contrarotative cells are formed as shown in Figures To show the effect of the thermal conductivity ratio Kr on the thermal fields and the circulation of the fluid in the enclosure, the isotherms and streamlines are presented in D = 0.3 and Kr = 5. As can be seen in Figures D and thermal conductivity ratio, Kr = 1. The result presented in D = 0.3. Obviously, the heat transfer increases by increasing Ra. The heat transfer enhancement by increasing Ra is more pronounced at higher values of thermal conductivity ratio as shown in 5. This figure shows that the heat transfer decreases by increasing the solid wall thickness D. There is a considerable difference between the heat transfer for small and large values of Kr. For Kr = 0.5, the heat transfer is almost constant. When Kr takes higher values, heat transfer drops sharply by increasing D and becomes a conduction mode. This is due to the bottom solid wall that behaves as an insulated material in this case. Finally, the average Nusselt number can be correlated pretty well with the wall thickness, the thermal conductivity ratio, and the Rayleigh number as follows:Variations of the average Nusselt number with the Rayleigh number are shown in 4 ≤ Ra ≤ 106, 0 ≤ D ≤ 0.4, and 0.5 ≤ Kr ≤ 10. Note that the maximum error is less than 5% for the valid ranges.This correlation is valid for 5 × 10The strength of the flow circulation of the fluid is much higher with thin walls and/or higher value of the solid to fluid thermal conductivity ratio.The number of contrarotative cells and the strength of the circulation of each cell can be controlled by the thickness of the bottom wall, the thermal conductivity ratio, and the Rayleigh number.The average Nusselt number increases by increasing either the Rayleigh number and/or thermal conductivity ratio, but the average Nusselt number decreases by increasing the wall thickness.The present numerical simulations study the effects of conduction in bottom wall on Rayleigh-Bénard convection in a square enclosure. The dimensionless forms of the governing equations were solved using the finite difference method. Detailed computational results for flow and temperature fields and the heat transfer rates in the enclosure have been presented in graphical forms. The main conclusions of the present analysis are as follows.


The Funding statement in the PDF version of this article is incorrect. The publisher apologizes for the error. The correct Funding statement is: This work was supported by funding from the Cancéropôle Lyon Auvergne Rhône Alpes and the French NCI (under PHRC grant 12007 and Lyric grant INCa-DGOS-4664). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


In the Funding section, the grant number NNJ10ZSA001N from the funder National Aeronautic and Space Administration has been updated by the funder. The updated grant number is: NNX11AD22G.