Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of  The discovery of regulatory RNA has contributed to a paradigm shift in the field of molecular biology . PreviouBriefly, siRNAs are ∼21–24 nucleotide (nt) molecules generated from double-stranded RNA precursors. siRNAs act on RNA molecules by initiating canonical RNA degradation, or upon DNA molecules via RNA-directed DNA methylation . siRNAs Advances in sequencing technology have facilitated the discovery of vast populations of sRNAs, many of which are developmentally regulated . Similarin vitro methodologies such as 5′ RACE (Agrobacterium tumefaciens infiltration assay (agroinfiltration) on Nicotiana benthamiana leaves, and subsequent molecular analyses have been used to study miRNA-target gene interactions  assay was applied to study complementarity requirements for miRNA-target interactions using the transient agroinfiltration assay on a leaves . In the a leaves . FurtherN. benthamiana leaves with the quantitative dual LUC assay system. We report the minimum leaf extract dilution required to avoid inhibition of the firefly LUC reporter. We also report the optimal ratio of miRNA precursor construct to reporter construct and the minimum incubation period post-agroinfiltration to maximize miRNA directed knockdown of target gene expression. Additionally, we describe improved expression of the Renilla LUC reporter under the control of a tomato ACTIN promoter, compared to the strongly wound induced but relatively weak expressing A. tumefaciens NOS promoter. We applied the assay to validate computationally predicted targets of rice miRNA529b with just six replicate samples, demonstrating the scalability of the optimized assay conditions. Considerations for experimental design, current limitations, and extended applications of the assay are discussed.This study investigates key parameters and experimental conditions required to maximize miRNA-directed repression of a target sequence when using a transient assay that combines co-agroinfiltration of SalI restriction enzyme (New England Biolabs) followed by T4 DNA polymerase (New England Biolabs) treatment and self-ligation, in order to inactivate the SalI restriction site between the NOS promoter and the Renilla LUC coding sequence (verified by Sanger sequencing of the NOS promoter-Renilla LUC coding sequence junction). The resulting intermediary plasmid was then double digested with EcoRI and PstI (New England Biolabs), followed by Antarctic Phosphatase treatment (New England Biolabs). An adaptor containing a SalI restriction site was made by annealing two complementary primers with 5′ phosphate additions and EcoRI and PstI overhangs. The adaptor was subsequently ligated into the intermediary plasmid to construct pGrDL_SP .The plasmids used in this study were constructed by modifying the pGreen dualLUC 3′ UTR sensor plasmid described by NOS promoter in pGrDL_SP was replaced with a tomato ACTIN promoter to construct plasmid pGrDL_SPb . This was achieved via PCR amplification of the tomato ACTIN-7 promoter  using a 5′ primer containing an FspI restriction site and a 3′ primer containing a SalI restriction site . Approximately 10 nmol of each primer were mixed together in a volume of 60 μL of DNAse-free water, heated to ∼90°C for 5 min in a water bath and allowed to slowly cool to room temperature (at a rate of ∼1 degree per minute). The resulting adaptors were diluted 50-fold prior to directional cloning into a SalI and PstI digested and Antarctic Phosphatase (New England Biolabs) treated pGrDL_SPb plasmid, and subsequently verified by Sanger sequencing .Target site adaptors were made by annealing two complementary primers (Supplementary Table www.mirbase.org) was synthesized with flanking restriction enzyme sites (GenScript) and cloned between the double Cauliflower mosaic virus (CaMV) 35S promoter and 35S terminator of the expression plasmid pKENminusbar . A similar sized construct, derived from Capsicum chlorosis virus, was cloned into pKENminusbar and used as a control. This control sequence (Supplementary Table The rice miRNA precursor for miR529b (accession MI0005804 from A. tumefaciens strain GV3101 (harboring pSOUP) and plated on LB agar media with rifampicin (25 μg/mL), kanamycin (50 μg/mL), and tetracycline (10 μg/mL) selection. Starter cultures were prepared by inoculating a single colony in 2 mL of LB with rifampicin, kanamycin and tetracycline selection, and grown overnight at 28°C in a shaking incubator. Starter culture was used to inoculate the working culture , which was grown overnight under the same media, selection and incubation conditions. Working cultures were harvested by centrifugation at 24°C and 2,400 × g for 15 min. Cell pellets were resuspended in 10 mM MgCl2 and the OD600 adjusted to 0.5 prior to the addition of acetosyringone (200 μM). The resulting cell cultures were stored in the dark at room temperature for either 4 or 24 h prior to infiltration.Expression plasmids were transformed by electroporation into Nicotiana benthamiana plants were grown at room temperature and 16 h light per day, under LumiBar LED strip lighting (LumiGrow) intensity setting four. Seedlings were grown for approximately 3–4 weeks prior to agroinfiltration. Three expanded leaves per plant were infiltrated by applying pressure on the abaxial surface of the leaf with a disposable 5 mL syringe containing the Agrobacterium suspension. Each leaf was treated as a biological replicate. Agroinfiltrated plants were incubated for a specified period  in a growth chamber set to 22°C with 16 h light per day. Agroinfiltrated leaves were harvested individually, snap-frozen in liquid nitrogen, powdered using a ball mill tissue-lyser (Retsch), and stored at -80°C prior to measurement in dual LUC assays.g for 1 min. The supernatant was typically diluted 20-fold in PLB and 15 μL loaded into a well of a white flat bottom Costar 96 well plate (Corning). The assay was performed using a GloMax 96 microplate luminometer (Promega). The dual injectors were used to introduce 75 μL of luciferase assay reagent and Stop & Glo reagent, respectively, per well. Luciferase assay reagent and Stop & Glo reagent components are provided in the Dual Luciferase Reporter Assay System kit (Promega).Dual luciferase (LUC) assay extracts were prepared using the Dual Luciferase Reporter Assay System kit (Promega). Typically ∼5 mg of powdered tissue was mixed with 100 μL of the passive lysis buffer (PLB) provided in the Dual Luciferase Reporter Assay System kit, and the cellular debris pelleted by centrifugation at 7,500 × Statistical analysis of the resulting data was performed using GraphPad Prism 6 software.N. benthamiana leaf extracts were capable of inhibiting firefly or Renilla LUC activity and what dilution factor, if any, is required to negate the inhibition of activity. Extracts were prepared from N. benthamiana leaves agroinfiltrated with the dual LUC plasmid pGrDL_SPb  at a starting concentration of 5 mg per 100 μL of PLB. LUC activity measurements revealed substantial inhibition of firefly LUC activity in undiluted extracts . The level of inhibition varied substantially from sample to sample, with LUC activity levels between 26 and 49% of that in diluted samples. In contrast, inhibition of Renilla LUC activity was minimal in the undiluted leaf extracts . The inhibition effect on firefly LUC was lessened by dilution, plateauing out after 20-fold dilution . Thus we recommend preparing at least 20-fold dilutions of extracts made from no more than 5 mg leaf tissue per 100 μL PLB to negate any inhibitory impact on firefly LUC activity. All subsequent optimisation experiments presented in this article utilized 20-fold dilution of extracts containing < 5 mg leaf tissue per 100 μL PLB.Previous studies in sugarcane revealed that undiluted plant extracts can inhibit firefly luciferase activity . We firsSalI and PstI restriction sites. This modified plasmid was named pGrDL_SP  . The Renilla LUC expression cassette in pGrDL_SP is driven by the NOS promoter. It is reported that NOS promoter activity can be strongly induced by wounding in leaf tissues (NOS promoter yields an order of magnitude fewer relative light units (RLUs) than the firefly LUC expression cassette under the control of the CaMV 35S promoter. To address these concerns, we substituted the NOS promoter with a tomato ACTIN promoter sequence to form plasmid pGrDL_SPb . We then compared the RLU yield from the Renilla LUC reporter generated by each plasmid after agroinfiltration of N. benthamiana leaves.The pGreen dualLUC 3′ UTR sensor plasmid described by  tissues . As agroACTIN promoter drove substantially higher Renilla LUC expression on average than the NOS promoter, thereby increasing the sensitivity of the assay . Accordingly, the RLU values generated from the tomato ACTIN-Renilla LUC expression cassette were closer to those obtained from the CaMV 35S-firefly LUC expression cassette, resulting in lower dual LUC ratios generated from pGrDL_SPb . The reduced interquartile range of the dual LUC ratios generated from pGrDL_SPb indicates the replacement of the NOS promoter with the tomato ACTIN promoter may have also reduced variability. The dual LUC plasmid pGrDL_SPb was used in all further optimisation experiments.The tomato CaMV 35S promoter in expression plasmid pKENminusbar . Rice miR529b is evolutionarily related to miR156  complementary sequence to rice miR529b was selected as the target sequence and cloned into the 3′ UTR of the firefly LUC construct within the dual LUC reporter plasmid pGrDL_SPb. Although the 3′ and central seed regions of miR156 overlap the targeted sites of miR529b, it is unlikely endogenous miR156 could effectively target the cloned miR529b target sequence due to the absence of target sequence complementary to the important 5′ seed region . As a consequence, all further optimisation steps presented in this article used a 10:1 ratio of miRNA precursor culture to target in dual LUC reporter pGrDL_SPb culture.A previous study used a 1:1 ratio of miRNA precursor plasmid  culture . To invet-test . A strong and highly significant knock-down of expression  was detected 3 days post-agroinfiltration . A similar if not greater knockdown of expression was found at 4 and 5 days post-agroinfiltration. However, the raw RLU values peaked at day 3 and declined from day 4 to day 5. Therefore we recommend an incubation period of 3 days post-agroinfiltration.To assess the optimum incubation period for miRNA-directed knockdown of reporter containing the target sequence, agroinfiltrated leaves were harvested each day for 5 days post-infiltration. The expression of the Renilla LUC reporter was near background at day 1, and therefore no data was analyzed at that time point. Both the firefly LUC and Renilla LUC raw values were relatively low at 2 days post-agroinfiltration, and the dual LUC assay ratio indicated a minor knock-down effect that did not pass an unpaired Agrobacterium culture in order to induce the virulence genes. However, reported studies vary in the duration of this incubation period, ranging from 1 to 24 h .Agroinfiltration protocols specify addition of acetosyringone to the  to 24 h . To asseNOS promoter to drive Renilla LUC expression, whereas this study used the relatively strong tomato ACTIN promoter. Further, Agrobacterium culture to target plasmid Agrobacterium culture, incubated their plants for just 2 days post-agroinfiltration, and did not apply a dilution factor to their plant extracts prior to running the dual LUC assay. We hypothesized the optimisation described in this study would reduce variability and enhance the level of miRNA-mediated repression of target gene sequence constructs. A direct comparison of the two methods was undertaken pairing rice miR529b precursor with the known target sequence from OsSPL14 cloned into either pGrDL_SPb or the pGreen dualLUC 3′ UTR sensor plasmid described by Figure 7). Importantly, variability was lower in the optimized assay as evidenced by the reduced inter-quartile range .A previously published study used a dual LUC assay to quantify miRNA-mediated repression of targeted gene sequences . FollowiN. benthamiana leaves and 20-fold dilutions of ∼5 mg/100 μL leaf extracts were used to experimentally validate computationally predicted targets. The psRNAtarget online resource was used to screen for rice transcript sequences that are computationally predicted targets of rice miR529b . The strength of the repression effect varied between the different target constructs. The highest scoring predicted target sequence was from transcript OsSPL14 and this target construct generated the strongest repression effect. Lower scoring predictions, including target sequences from transcripts OsHXT1;4 and OsFBX292, produced relatively subtle repression of reporter expression. Although the sample size for each treatment was small (n = 6), the repression effect in each test case was significantly different to the control treatments using a simple unpaired t-test. Two target sequences (named mismatch1 and mismatch2) containing little complementarity to miR529b were also tested. No difference in expression level was observed when these control target sequences were co-infiltrated with the miR529b precursor or the control precursor, indicating the miR529b-dependent repression of predicted target sequences was not due to a secondary effect.The optimized assay conditions of 10:1 miR529b precursor to reporter-target expression cultures including 3 days incubation post co-infiltration of  miR529b . Five of miR529b . The subn = 6), while the OsSPL14 and OsFBX292 target constructs generated average dual LUC ratios of 1.013  and 2.870 , respectively, in the control treatments. These constructs only vary by the sequence composition of the short target sequence inserted into the 3′ UTR of the firefly expression construct, yet these differences are sufficient to cause noticeable differences in the stability of the reporter gene expression in the controls. This variability between different test constructs highlights the need for appropriate controls. Previous reports have compared test constructs to a control construct containing a non-targeted sequence, assuming the stability of reporter expression is not affected by the different target sequences  and RNAi constructs (data not shown). Artificial microRNA (amiRNA) refers to a technique whereby the mature miRNA sequence of a natural precursor miRNA is replaced with an artificial mature miRNA sequence designed to target an RNA transcript of interest . HoweverIt is also likely the assay can be extended to assess the ability of other sRNA classes to repress expression of putative target sequences. For example, interactions of sRNAs generated from plant pathogens with complementary plant target sequences could be assessed by comparing the dual LUC ratios generated in infected vs. non-infected tissues. However, assessing target interactions of mobile classes of sRNAs may require an amendment to the protocol where agroinfiltration should occur on just one leaf per plant.N. benthamiana as the host plant. miR529b is not endogenous to N. benthamiana, and therefore we were able to reduce the likelihood of endogenous miRNAs interacting with the target sequences. Using the assay to assess miRNA-target interaction when the miRNA is endogenous to N. benthamiana can be problematic, as we have found that reporter-target repression due to abundant endogenous miRNA present in control plants co-agroinfiltrated with a non-functional precursor can be just as severe as in the test plants co-agroinfiltrated with the overexpression miRNA precursor construct (data not shown). This hurdle could be overcome by modifying the control strategy. In such cases, agroinfiltration of the target plasmid could be directly compared to agroinfiltration of a mutated version of the target plasmid to reveal endogenous miRNA-target sequence interaction. However, the possibility of introducing mutations that differentially affect transcript stability cannot be discounted.Limitations of the assay include difficulties encountered in assessing miRNA that are endogenous to the host plant. In this paper we applied the optimized assay to assess the interaction of rice miRNA529b with putative rice targets using There is scope to further improve the assay by, for example, using codon-optimized versions of the firefly LUC and/or Renilla LUC to improve sensitivity . The morThis study addresses the challenge of experimentally validating large numbers of computationally predicted transcript sequences that might be targeted by complementary miRNA. The study builds on the use of the dual luciferase assay in combination with agroinfiltration transient expression protocols, by optimizing experimental design and key parameters. The transient basis of the assay allows for a rapid assessment of miRNA action on a target sequence without the need to generate transgenic lines. The quantitative power of the dual LUC assay allows detection of relatively subtle miRNA-target sequence interactions. The optimisation of the assay and careful experimental design allows observation of statistically significant differences in reporter expression from as few as six replicate samples, thereby providing scalability. The assay is versatile and could be applied to observing a number of small RNA-target interactions.RM and PS designed the experiments. RM and LC cloned the constructs. RM, LC, L-SP, GS, and EN performed the dual LUC assays. JD-M performed computational analysis to predict miR529b target sequences. RM and LC analyzed the data. RM drafted the manuscript and all authors edited and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding the combustion behavior of gel fuel droplets is pivotal for enhancing burn rates, lowering ignition delay and improving the operational performance of next-generation propulsion systems. Vapor jetting in burning gel fuel droplets is a crucial process that enables an effective transport (convectively) of unreacted fuel from the droplet domain to the flame zone and accelerates the gas-phase mixing process. Here, first we show that the combusting ethanol gel droplets (organic gellant laden) exhibit a new oscillatory jetting mode due to aperiodic bursting of the droplet shell. Second, we show how the initial gellant loading rate (GLR) leads to a distinct shell formation which self-tunes temporally to burst the droplet at different frequencies. Particularly, a weak-flexible shell is formed at low GLR that undergoes successive rupture cascades occurring in same region of the droplet. This region weakens due to repeated ruptures and causes droplet bursting at progressively higher frequencies. Contrarily, high GLRs facilitate a strong-rigid shell formation where consecutive cascades occur at scattered locations across the droplet surface. This leads to droplet bursting at random frequencies. This method of modulating jetting frequency would enable an effective control of droplet trajectory and local fuel-oxidizer ratio in any gel-spray based energy formulation. In this light, there has been a considerable interest in high-performance gellant based fuels as alternatives to conventional neat propellants10. This is due to their enhanced rheophysical properties that syndicate the advantages of both solid and liquid fuel propellants. For instance, a solid-like behavior at low or negligible shear reduces the chance of a hazard due to accidental spill or leakage (particularly in case of toxic and hypergolic propellants), thus, facilitating a safe handling and storage11. High viscosity during storage also enables a stable and homogenous suspension of energetic nanoscale additives in the fuel matrix which increases the fuel energy density and specific impulse of the engine. On the other hand, liquid-like behavior at high shear offers the flexibility of thrust modulation and re-ignitability in engines. Furthermore, the shear thinning property ensures that the gelled fuels can be pumped easily into the combustion chamber like liquid propellants and aid an effective atomization when forced through injectors17.The success of future rocket propulsion systems will depend on their ability to utilize eco-friendly fuels that exhibit high exothemicity, shortened ignition delay time, high energy density and operational safety at low cost (i.e. easy to store and handle)18 that occurs asymmetrically and randomly during its lifetime. A typical jetting event involves four key stages that occur sequentially and at spatially distinct locations19. First, gellant crust/shell formation due to phase seperation of gelling agent from the base fuel/gellant solvent (near the droplet surface). Second, boiling of trapped fuel (inside the droplet). Third, bursting of the gellant shell due to internal pressure build-up and jetting of unreacted fuel vapors (across the droplet-gas interface) and fourth, jet travel to the flame envelope: which causes distortion in the symmetric tear-drop shaped flame geometry.In spite of these potential advantages, gel fuels exhibit complex combustion and rheological characteristics compared to solid/liquid propellants. For instance, disruptive burning of gel fuels is highlighted by jetting of unreacted fuel vaporsjetting and microexplosion have been used interchangeably in the existing literature for highlighting disruptive burning of gelled fuel droplets. However, they represent a completely different class of disruption events. As first reported by Law22, microexplosion corresponds to a sudden catastrophic break-up of the droplet that occurs due to rapid internal gasification of high volatility component. Specifically, the low boiling point species is diffusionally entrapped in the droplet core and gets superheated to its homogenous boiling limit, thereby, resulting in an immense pressure build-up and catastrophic breakup of the droplet. This occurs only once and marks an end to the droplet lifetime. Unlike microexplosion, jetting events occur throughout the droplet lifetime as bubbles form, grow and collapse continuously to expel out the unreacted fuel vapor. Such continuous bubble formation and existence of a large bubble population is typical of heterogeneous boiling. Since heterogeneous boiling features a low degree of stored superheat the jetting events have a noticeably low intensity as opposed to a microexplosion.It is noteworthy to consider that the terms vapor jetting to a certain degree is similar to the bubble ejections observed during burning of colloidal fuel droplets27 since latter also features multiple bubble formation. However, in terms of the thermo-physical processes governing their initiation, growth, termination and their post-termination effects on the combustion behavior, vapor jetting and bubble ejections are markedly different disruption modes. Firstly, in case of nanofluid fuel droplets the nanoparticles (NPs) agglomerate to form micro scale aggregates which then act as nucleation sites23. In contrast, in gel fuel droplets it is the inner surface of viscoelastic gellant shell that serves as a nucleation surface. Secondly, in nanofuel droplets the internal accumulated pressure is released through rupture of the crust that is formed by the consolidation of nanoparticles at the receding droplet-gas interface. This rupture accompanies the formation of daughter droplets (DDs) that pinch-off from the parent droplet24 and may further undergo puffing and microexplosion30. Contrarily, in gel fuel droplets the rupture of gellant shell is not accompanied by the formation of any DDs but results in jetting out of unreacted fuel vapors through ruptured portion of the shell, thereby releasing the internal pressure. Thirdly, in nanofuel droplets the shell formation process is governed by coupled but competing mechanisms of NP agglomeration and secondary atomization that alter the mass fraction of NPs within the droplet dynamically. The former aids shell development through particle aggregation while the latter tends to disrupt the shell directly through rupture and indirectly by continuous efflux of NPs26. However, in gel fuel droplets the shell formation occurs through phase separation of the gellant35 from the base fuel/gellant solvent/gellant formulation and the whole gellant mass is retained within the droplet domain until it carbonizes and gets consumed towards the end of droplet lifetime. Finally, in nanofuels and in general in other multi-phase multicomponent droplets, the formation of DD’s (secondary atomization) and their subsequent break-up (tertiary atomization)29 facilitates an enhancement in burn rate by increasing the net surface area and distributing the fuel charge uniformly. Additionally, the DD’s act as carriers through which the energetic nanoparticles present within the droplet domain are transported to the flame to harness their energy27. However, the significance of jetting observed in case of gel fuel droplets lies in its ability to carry the unreacted fuel from the droplet to its surrounding. This enhances the fuel mass flux, thereby, facilitating faster burn rates. This is particularly crucial for rocket motor applications utilizing gel propellants since gels exhibit a higher ignition delay and low burning rates due to their inherently high heat of vaporization36. By utilizing high-speed OH-PLIF in burning monomethyhyrazine (MMH) droplets (gelled with Hydroxypropyl cellulose HPC), Cho et al.37 illustrated that such disruptive burning exhibits a tri-modal behavior. First, the jets may corrugate the flame envelope. Second, the jets may break flame envelope  and, third, jets form a local fireball outside the flame envelope. The occurrence of a particular disruptive mode depends on the jetting intensity (i.e. maximum jet speed) which is highest for mode 3 (~0.8 m/s) and lowest for mode 1 (~0.35 m/s). Previous studies have shown that the jetting velocity can be altered by varying initial droplet diameter38, regulating external ambient conditions38 and varying the functional properties such as the parent fuel, initial gellant concentration/type (organic or inorganic gellants)39 and through addition of energetic nanophase additives . Literature search reveals that most studies have focused on jet visualization at the flame scale while the identification of different jetting modes, their initiation mechanisms and methods to control them at the droplet scale has received little attention.With context to heterogeneous boiling, oscillatory jetting in a reacting environment due to acyclic bursting of its shell. (2) By varying the initial gellant loading (3 to 6 wt.%) the bursting frequency can be modulated and (3) by applying set-theory to the rupture jet holes (in the shell) different jetting states of the droplet can be identified i.e. coincidental, off-set or displaced.From a viewpoint of combustion efficiency organic gellants offer several advantages compared their inorganic counterparts: First, unlike inorganic gellants that are inert the organic gellants are consumed completely during the burning process and hence they contribute to the heat of combustion with no left over residue. Secondly, inorganic gellants aid in the formation of a rigid, impervious shell which eventually leads to droplet microexplosion, thereby, displaying an uncontrolled atomization. In contrast, organic gellants form a viscoelastic shell that ruptures and re-establishes as jetting events proceed. This offers an opportunity of modulating the jetting characteristics (intensity and frequency) by tuning the shell properties simply by varying the gellant concentration. This is particularly attractive since it offers an ease of control at the initial stage of fuel formulation. We exploit this behavior to show that: (1) an eco-friendly ethanol gel droplet containing a cellulose based derivative as the gelling agent (Hydroxy methyl Propyl Cellulose: HPMC organic gellant) displays 43 in liquid rocket engines. Interestingly, previous studies have shown that the pure fuel  droplet flames are also susceptible to oscillations when subjected to external acoustic excitation either through travelling44 or standing pressure waves43. In the latter case, an increase in droplet burn rate by as large as ~15% under normal gravity conditions has been reported when the sound pressure level exceeded ~135 dB and droplet was placed near pressure node. On the contrary, there was a little observable change in gasification rate when droplet was stationed at velocity node. Further, the droplet diffusion flames were found to be most responsive at low excitation frequencies (~5–330 Hz)44 while they exhibited diminished response at higher forcing frequencies (>400 Hz). This indicates that oscillation mechanisms in combusting droplet exhibit multi-parameter dependence and hence provide an innovative means of manipulation at the droplet scale, which form a sub-grid elements of any gel spray based combustion strategy.A fundamental understanding of oscillatory droplet combustion is also crucial from an application viewpoint given its relevance to combustion instabilitiesoscillatory rupture cascade. Such cascades occur intermittently during the intermediate stages of droplet lifecycle and each cascade constitutes a series of rupture cycles that occur aperiodically. Further, as shown in Fig. four stage process of: (1) Gellant layer rupture at a weak soft spot. This is a key stage that results in the hole formation and provides an opening for the outflux of unreacted fuel vapors via jetting. (2) Continuous jetting alongside expansion of the rupture site. (3) Continuous jetting alongside shrinkage of the ruptured site; eventually leading to the recovery of ruptured layer and (4) bubble growth period, which features an internal pressure build-up and initiation of next rupture cycle. Clearly, the last stage corresponds to a dormant or inactive period  and the thickness (δ) that vary with GLR. A physical analysis of the shell bursting dynamics is discussed in Supplementary information. Note that it is extremely difficult to predict the spatio-temporal variation of shell properties in a reacting environment where frequent ruptures are altering the shell dynamically. In this light, the rheometric measurements of gel fuel properties under isothermal conditions can provide critical insights on the oscillatory bursting behavior of droplets. For instance, Table strong but rigid shell formation i.e. high weak but flexible shell formation i.e. low During a cascade, the tendency of droplet bursting at any given time instant is governed by a direct competition between the ebullition activity that tends to scale-up internal pressure and the ability of viscoelastic shell to sustain this pressure upsurge. Before the shell ruptures it restrains pressure rise through stretching, thinning down and yielding as shown in Fig. mentclass2pt{minimsis Fig.  that preO (1.5 mm/s); O(1000 ms) i.e. comparable to the droplet lifetime. Besides, just prior to rupture the bubble grows to a markedly high droplet void fraction (~0.7) which causes severe droplet swelling   is preferentially vaporized and the gellant concentration at the droplet surface reaches a critical concentration (~0.9–0.95)35. This signifies that the droplet core would always be at a lower gellant concentration compared to the shell35. Therefore, following a high intensity rupture, the shell gets thinner in the ruptured region as this region is exposed to fresh gel from the droplet core while it gets thicker in the surrounding regions where the retracting sheet overlaps with the previously intact layer. In this new state, the ruptured region becomes a weak soft part in an otherwise strong crust and acts as a favored site for cascades. Formation of a weak region alongside jetting hole is shown in Fig. In contrast, at 5 wt.% Fig. , bubble 35 where a uniform shell formation was reported during combustion of gel fuel droplets. The jet hole depth is used to estimate the shell thickness δ averaged over multiple points along the circumference and found to be ~30 ± 15 µm and ~75 ± 20 µm at 3 wt.% and 5 wt.% GLR respectively. Also, average δ determined from crossectional view of the hollow precipitate for 5 wt.% case . Thus a higher GLR leads to a thicker shell on an average.Additionally, SEM image of cut-section through the final precipitate Fig.  illustraFurthermore, since high intensity bursting is driven by transient pressure spikes it tends to occur randomly across the droplet surface. This creates an entirely distinct and new shell configuration with each rupture. Given the complexity of shell rupture-reformation process at 5 wt.%, the probablity of randomly distributed oscillatory jetting sites across the droplet surface is quite high. Indeed, SEM images of Fig. i while the rupture cycles will be denoted by CAS i C j, where i and j = 1, 2, 3… represent the indices for N is total number of cycles in a given cascade) by an order of magnitude i.e. from Temporal fluctuations in circumferential strain  that have significantly short active time periods O (5 ms). However, the novel oscillatory jetting cascades observed in this study persist for a majority of droplet lifetime ~0.1–0.75 N) range from ~2–12, with the corresponding total active jetting time period ~O (1 s). Thus, given the long durations of active jetting during cascades, they contribute majorly to the efflux of unreacted fuel vapor mass from the droplet domain and hence represent the most critical mode of fuel vapor transport. Previous studies39 also postulated that jets emanate repeatedly along the same angular direction. In this context, the relative spreading factor Currently, significant effort is directed in understanding the combustion behavior of gel fuel droplets with an aim of enhancing burn rates and energy density of traditional hydrocarbon fuels. This is done either by regulating the ambient conditions or by varying functional properties such as initial droplet diameter, base fuel and gellant type/concentration. However, jetting of fuel vapors is a key phenomenon in combusting gel droplets that has received little attention. In addition to vaporization, jetting enables an advective transport of unreacted fuel vapors from the droplet to the flame. This increases the fuel efflux and shortens F/O mixing timescales, thereby, promoting efficient combustion. In the existing literatureKey findings indicate that by varying the initial gelling loading rate (GLR) the bursting frequency of the droplet (or jetting frequency) can be altered. At high GLR, a thick-strong-rigid shell is formed where successive cascades occur at randomly dispersed locations on droplet surface and lead to droplet bursting at random frequencies. However, at low GLRs, a thin-weak-flexible shell is formed that is prone to consecutive rupture cascades in same region of the droplet. This leads to droplet bursting at increasingly higher frequencies. Thus, this method of tuning jetting frequency serves a two-fold purpose. First, homogenizing the fuel charge both by altering the droplet trajectory and enhancing the vapor-phase mixing and second, tuning the burn rates through a controlled release of fuel vapors. Since several industrial processes ranging from microfluidics to pharmaceutics to lab-on-a-chip devices utilize gel droplets, our experiments may act as a versatile platform in understanding the fundamental mechanisms in gel based systems.In present experiments, a non-metallized ethanol gel containing organic gellant is used as the test fuel. Gelled ethanol consists of three parts: 1) Research grade ethanol  which is the base fuel, (2) macromolecular Hydroxypropyl Methyl Cellulose  which is the actual organic gellant (OG) and (3) double distilled water that acts as a base solvent for the OG. All chemicals were procured from Sigma-Aldrich Co. Table  Researchµ-syringe with a 30-gauge ID needle is used to dispense a ∼2.8 µl gel fuel droplet at the end of 80 µm fused quartz fiber. Due to small fiber diameter (less than 100 µm) and low thermal conductivity (1.4 W/m-K at 293 K) of fused quartz any physical or thermal interference due to the suspension fiber can be neglected45. Droplet ignition is achieved through a 150 µm Nichrome wire. The heat input required to achieve droplet ignition (i.e. the ignitor operation time) is regulated by a DC electric power source. The combustion behavior of ethanol-gel droplets in pendant mode is investigated at ambient temperature and pressure conditions and under normal gravity.A volume calibrated An ultra-high speed Photron FASTCAM SA-X2 camera attached with a 6.5 X Navitar Zoom lens is used to record the bursting dynamics of an Ethanol/Water/HPMC droplet at 20,000 fps (Exposure time: 60 µs) and at a spatial resolution of 3.8 µm/pixel. Since it is difficult to form spherical droplets with gel fuels, the initial droplet diameter (Supplementary InformationSupplementary Video 1Supplementary Video 2


Objective Nontraumatic acute cervical disk herniation resulting in acute severe neurologic deficit is a rare entity described in a limited number of case reports. We describe the management and outcome in patients presenting with severe neurologic deterioration caused by acutely herniated cervical disks.Methods Four patients (mean age 39.5 years) presented to our tertiary care academic medical center from September 2012 to September 2013 with severe progressive neurologic deficits due to cervical disk herniation and were included in the series. Patients' surgical, medical, and imaging records were retrospectively reviewed under an Institutional Review Board waiver of informed consent.Results Patients in the series presented with acute neurologic deterioration, including paraparesis, Brown-Séquard syndrome, or quadriparesis deteriorating to quadriplegia. Emergent magnetic resonance imaging (MRI) scans and emergent decompression and fusion for acute soft disk herniation were performed in all cases. All patients recovered to excellent functional status with Frankel score improvement from B (one patient)/C (three patients) to E (three patients)/D (one patient).Conclusions Acute cervical disk herniation with acute neurologic deterioration is a medical emergency necessitating emergent MRI and surgical decompression. Clinical presentation varies. In patients with rapid-onset neurologic deterioration, a high level of suspicion for this rare entity is indicated. Cervical disk disease usually causes cervical myelopathy or radiculopathy with a gradual stepwise neurologic deterioration.We report presenting symptoms, treatment, and outcomes of patients with acute cervical disk herniation causing acute neurologic deteriorations treated in our medical center.Four patients  presented to our tertiary care academic medical center from September 2012 to September 2013 with progressive paresis due to cervical disk herniation and were included in the series. The patients' surgical, medical, and imaging records were retrospectively reviewed under an Institutional Review Board waiver of informed consent.A 34-year-old otherwise healthy man presented to the emergency room with quadriparesis. Three weeks prior to his admission, he had experienced neck pain and limb numbness. Physical examination revealed upper and lower extremity weakness (4/5), sensory level at T2, and hyperreflexia in all four limbs with clonus and positive Babinski sign in the lower extremities. Emergent cervical T2-weighted MRI revealed a huge C5–C6 herniated disk with spinal cord compression . Repeatamwith right hemiparesis. His medical history was unremarkable, although he had complained of neck pain 1 day before admission. Physical examination revealed severe hemiparesis of the right upper and lower extremities  and sensory disturbances on the left side (diagnosed as Brown-Séquard syndrome). Contrast-enhanced brain computed tomography (CT) and CT angiography were normal. Emergent cervical T2-weighted MRI revealed a C3–C4 right-sided disk herniation with marked spinal cord compression  and hyperreflexia in all four extremities with positive Babinski sign and a sensory level at T6 (Frankel grade C). Emergent T2-weighted MRI of the cervical and thoracic spine demonstrated a degenerated C5–C6 disk with herniation resulting in slight spinal cord compression; however, a huge C6–C7 herniation was seen with cranial disk migration and severe cord compression . DuringA 47-year-old woman presented to the emergency department complaining of cervical pain and inability to stand or walk. She had a history of Crohn disease, fibromyalgia, major depression, drug abuse, and borderline personality disorder. Independent examinations by a neurosurgeon, neurologist, and orthopedic surgeon produced concurring assessments of slight limb weakness, inconsistent with her complaints, and speculation that her difficulty in walking was of a nonorganic origin. A CT scan demonstrated C5–C6 disk herniation with spinal canal compromise . The paWe present our experience in the management of four patients with no history of recent traumatic injury who presented with acute neurologic deterioration due to cervical disk herniation. Based on findings of cord compression on MRI, emergent surgery was performed, enabling all four patients to achieve a normal gait at 2- to 3-month follow-up. All patients were treated with spinal arthrodesis because the authors prefer motion restriction rather than motion preservation for patients with myelopathy. It is noteworthy that all patients in this series presented to the emergency room with only a partial deficit and were treated emergently.Most acute cervical disk herniations are sequelae of traumatic injuries and are treated acutely as such. Degenerative spine disease is typically a chronic condition with gradual exacerbations. In 20 to 62% patients with cervical myelopathy, the condition deteriorates over 3 to 6 yearsAcute cervical disk presenting as acute Brown-Séquard syndrome is rarely described in case reports.Acute quadriplegia resulting from cervical herniated disk was described by Sadanand et al in a 42-year-old patient with acute presentation after a sneeze.In our series, patient 1 presented with quadriparesis that progressed to complete quadriplegia after admission MRI. The patient was operated within 3 hours of deterioration and recovered a normal gait and extremity strength. Perhaps because of her somewhat complex medical history, the walking difficulties of patient 4 were initially thought to be of a nonorganic origin despite examination by physicians from several subspecialties. Only after a rapid deterioration to Frankel grade C was the source of the problem correctly diagnosed. Fortunately, emergent surgery restored her neurologic function to near normal levels (Frankel grade D).All patients in this series had symptoms of neck pain with mild motor or mild sensory disturbances prior to their severe neurologic deterioration. The time frame for these symptoms ranged from 1 day to 3 weeks prior to admission. Possible causes for these early symptoms could be injury to the annulus fibrosus or disk herniation with slight pressure on the spinal cord. We could not find any red flags to differentiate these patients from other patients in whom disk herniation led to a more typical presentation of gradual myelopathy progression.All patients in this series underwent MRI examination within hours of the onset of marked neurologic deterioration, enabling urgent diagnosis and intervention. It is noteworthy that MRI may not be universally available on an emergency basis. The 2013 Organization for Economic Cooperation and Development report revealed that in 2011 the number of MRI scanners per capita varied widely, from 46.9/million population in Japan and 31.5/million in the United States to 10.8/million in Germany, 5.8/million in the United Kingdom, and only 2.5/million in Israel.Our study is limited by its retrospective nature and by the relatively small number of patients presented. These factors limit our ability to present statistical results to prove our hypothesis. However, to the authors' knowledge, it is the largest and only series of patients presenting with acute nontraumatic cervical disk herniation associated with spinal cord compression leading to sudden-onset rapid neurologic deterioration; thus, we believe the series emphasizes the importance of rapid diagnosis and intervention in this setting.Acute neurologic deterioration as a result of acute cervical disk herniation is a rare entity with variable presentation. The outcome in patients operated with incomplete deficits is better than surgery only after deterioration to a complete neurologic deficit; hence, rapid diagnosis and treatment are crucial. The capability to perform urgent MRI and surgery for patients presenting with neurologic deterioration should be mandatory for tertiary spine centers.


Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea.r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways.To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel’s The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding.The online version of this article (doi:10.1186/s12870-017-1080-9) contains supplementary material, which is available to authorized users. Pisum sativum L.), chickpea (Cicer arietinum L.), common bean (Phaseolus vulgaris) and lentil (Lens culinaris Medik.). The composition and nutritional qualities of pulses have been the subject of recent reviews , chain elongation [StSynII (rug5)] and branching [SBEI (r)].In this study, we focused on association mapping of variation in debranched starch CLD and %amylose with polymorphisms in 25 candidate genes representing 16 carbohydrate and starch metabolic enzymatic reaction classes Table . Using tn = 92) we employed, 2) the limited number (25) of candidate genes in the starch biosynthetic pathway that were characterised, and 3) that the candidate genes were partially sequenced, with a bias for intron-containing regions, therefore causal variants may have occurred outside the sequenced regions and may have been in only partial LD with variants within the sequenced regions. The genome-wide association study (GWAS) offers an alternate approach to the candidate gene-based approach for identifying genes associated with complex traits such as the composition of pea seeds, although GWAS is poor for detecting effects of minor alleles [The power of our study to identify sequence variants associated with starch CLD variation was limited. The limiting factors included: 1) the relatively small sample size  versus wrinkled (rr) lines. The mean proportion of intermediate length chains was increased in round seeded lines  also affected the mean proportion of intermediate length chains, which was higher on average for the lines with the G allele at that site.CLD affects gelatinization and pasting properties of starch , 9. As anes Fig.  with an r locus (SBEI), and a smaller effect of rb locus (AGPL1) mutations. Mutations in rug5 (StSynII) were also found to affect CLD in both developing and mature pea embryos [r, rb (AGPL1) and rug5 (StSynII) was associated with CLD variation (Table PGMP (rug3), GBSSI (lam) and SuSy (rug4), the other three genes identified, based on EMS mutations, in the above studies as being involved in starch metabolism.Mutational studies in pea have demonstrated the role of six pea seed starch biosynthetic genes in pea seed starch accumulation, structure and function , 59, 60.r locus, the major gene responsible for the difference between round and wrinkled (high amylose) pea lines, in both trials, as expected. In starch extracted from plots from the Field2011 trial, %amylose was also associated with SNPs in UPGase. For the GH2010 trial, %amylose was only associated with r locus. This likely reflects the greater variation in %amylose observed among round seeded lines in the Field2011 trial than the GH2010 trial, in which variation between round seeded lines was not significant. The effects of mutations in pea starch biosynthetic genes on %amylose have previously been determined, with rug3 (PGMP) and lam (GBSSI) reducing the % amylose content of starch compared with the wild type round seeded line, while rug5 (StSynII) and r (SBEI) mutants increased the percent amylose [p > 0.001) of amylose content with SNPs in amplified fragments of the AGPL1 (rb locus), GBSSI (lam locus) and SBEII genes. However, we did not detect association in %amylose involving SNPs in these genes. This difference that may be due to the germplasm used in the different studies or to the relatively low power of both studies due to the numbers of lines used.Variation in %amylose was associated with allelic variation in  amylose . In a re amylose  have fouSBEI (r locus) had the greatest effect on both amylose content and CLD, whereas in rice, polymorphisms in GBSS (Wx) had the greatest effect on amylose content, and SSII on gelatinization temperature [SBE3 having only a minor effect on gelatinization temperature. In maize, polymorphisms in a sucrose synthase gene (sh1), AGPL1 (sh2) and SBEIIb (ae1) were associated with amylose content and pasting properties [SSII and SBE associated with physicochemical properties, similar to the results for CLD in pea, but differed from pea in that association with GBSS was also found [As CLD and amylose content affect the physicochemical properties of starches, there are parallels, as well as contradictions, between our results and those of association mapping studies which looked at physicochemical properties of starches from other species. In pea, perature , with SBoperties , while iso found . Where tAGPS2, UGPase, StSynII), although only the mutation in StSynII resulted in a non-synonymous codon change  on protein function using PROVEAN software [UGPase gene, one of the significantly associated polymorphic SNPs occurred immediately upstream of a predicted intron acceptor cut site, hence may affect transcript splicing in lines with one or the other of the variants, a possibility that is able to be tested experimentally. Therefore, with the possible exception of the intron acceptor site variation in the UGPase gene, it is unclear whether any of the significantly associated polymorphisms identified are directly responsible for changes that would affect the CLD phenotypic variation, either through changes in coding or non-coding regions. Since only portions of the candidate genes were characterized for sequence polymorphism, it is quite possible that the significantly associated polymorphisms that were detected are in LD with the causal mutation(s) to be found in the gene regions that were not sequence characterized. It is also possible that the causal variants underlying the CLD traits do not occur in the candidate genes but in linked sequences. Our analysis of the decay of LD in this population has been limited to examining the LD within individual fragment alignments (where the maximum pairwise LD (r2) within fragments was 1.0 or nearly), which is of limited value because of the small size of these fragments, and to examining pairwise LD in pairs of fragments amplified from seven of the pea genes characterized  with UGPase (where the maximum pairwise r2 between genomic fragments from 59 to 201 and 1529–1601 on the mRNA was 0.833).Analysis of the gene context of the polymorphisms associated (FDR α ≤ 0.05) with CLD showed that most of these occurred in intronic sequences. For three of the genes, associated polymorphisms occurred in exonic regions  as having significant association with variation in %amylose. The findings were based on analysis of seed starch extracted from 92 diverse pea lines grown in two replicated trials, and on polymorphisms detected by sequencing fragments of 25 candidate genes and by scoring r locus. The r locus, which encodes SBEI, had the major effect (R2 of 83.4–88.6%), but other genes involved in substrate availability  and chain elongation (StSynII) were also associated with CLD variation, with effects ranging from 11.8 to 22.2% of the variation in mean peak area proportion at the most strongly associated peak, as determined by quantitative FACE. Examination of the sequence context of the significantly associated polymorphisms showed that most occurred in intronic regions, but a polymorphism in UGPase occurred immediately upstream of a predicted intron 3′ acceptor cut site, and a polymorphism in StSynII produced a non-synonymous mutation that was most likely to have a neutral effect on protein function. Hence, the candidate gene polymorphisms causing CLD variation in pea seed starch are in most if not all cases likely to be in full or partial LD with the associated polymorphisms that we have detected, and these causal variants may either occur within the candidate gene or in nearby sequences. Nevertheless, this study identifies sequence polymorphisms in carbohydrate and starch pathway genes, and publicly available pea lines containing the allelic variants, that can be used for further studies of genetic determination of pea seed starch structure and function, including plant breeding.We have shown that allelic variation in pea starch pathway candidate genes can have a measurable effect on amylopectin CLD. We identified polymorphisms in eight genes from the pea seed carbohydrate and starch metabolic pathway as having significant association (FDR ≤ 0.05 or FDR ≤ 0.10) with variation in debranched starch CLD, and two genes , r locus phenotype , % amylose and CLD mean molar peak area proportions for the Field2011 and GH2010 environments. (XLSX 136 kb)Pea single plant (PSP) accessions used for association mapping with Names and Collection Country from the USDA-ARS GRIN database  while the bottom panel shown correlations for n = 83 round seed lines only. (PNG 251 kb)Heat maps showing correlations between chain length distribution (CLD) mean peak area proportions for debranched starch from peas grown in GH2010 versus Field2011 trials. The colour scale ranges from blue (more strongly positive correlations) to yellow (more strongly negative correlations). The top panel shows correlations for all Additional file 3:2) within and among polymorphisms in 32 genomic fragments representing 25 candidate genes and r locus. Abbreviated candidate gene names are shown along the diagonal of the Figure. (PNG 56 kb)Heat map showing the extent of linkage disequilibrium  Principle components biplot for the first two PCs. Round seeded lines are indicated with clear circles and wrinkle seeded lines with red circles. (B) Scree plot of eigenvalues showing the variation in each component. (PNG 77 kb)Principle component analysis of population structure for Additional file 5:10(P) for the DP-environment combinations which gave associations with the lowest p-values. Results for the MLM + Q + K model (blue) and MLM + P + K model (red) are shown. The DP-environment combinations are indicated on the y-axes. (PNG 34 kb)Q-Q plots of the observed versus expected -log


Submarine mud volcanoes release sediments and gas-rich fluids at the seafloor via deeply-rooted plumbing systems that remain poorly understood. Here the functioning of Venere mud volcano, on the Calabrian accretionary prism in ~1,600 m water depth is investigated, based on multi-parameter hydroacoustic and visual seafloor data obtained using ship-borne methods, ROVs, and AUVs. Two seepage domains are recognized: mud breccia extrusion from a summit, and hydrocarbon venting from peripheral sites, hosting chemosynthetic ecosystems and authigenic carbonates indicative of long-term seepage. Pore fluids in freshly extruded mud breccia (up to 13 °C warmer than background sediments) contained methane concentrations exceeding saturation by 2.7 times and chloride concentrations up to five times lower than ambient seawater. Gas analyses indicate an underlying thermogenic hydrocarbon source with potential admixture of microbial methane during migration along ring faults to the peripheral sites. The gas and pore water analyses point to fluids sourced deep (>3 km) below Venere mud volcano. An upward-branching plumbing system is proposed to account for co-existing mud breccia extrusion and gas seepage via multiple surface vents that influence the distribution of seafloor ecosystems. This model of mud volcanism implies that methane-rich fluids may be released during prolonged phases of moderate activity. Therefore, terrestrial MVs represent a significant natural source of atmospheric methane with emissions estimated to range from 6 to 12.6 Tg/year6. The jointly expelled solids and fluids are referred to as mud breccia and over time the extrusion of mud breccia can build morphological edifices up to hundreds of meters in height that cover areas of up to several square kilometres7. Observations of terrestrial MVs suggest that mud volcanism is episodic, with periods of quiescence that may represent up to 95% of the lifetime of a MV, interrupted by brief eruptions (hours to days)8. Quiescent MV activity is typically characterized by the expulsion of mud and fluids from small cones (<10 m high) or fluid-mud pools and gas seeps8. These features may be linked to extensional faults within the extrusive edifice, in some cases defining sub-circular calderas thought to result from post-eruptive subsidence9.Mud volcanoes (MVs) are geological structures created by the extrusion of sediments, water, and volatiles (predominantly methane) from subsurface plumbing systems that may extend to depths of kilometres5) exceed that of onshore MVs by up to two orders of magnitude10. In the Black Sea, seepage from active MVs is associated with warm sediments ascending from depth and upward flow of aqueous fluids and free gas12. At the Håkon Mosby MV (Norway), variations in fluid flow have been found to trigger mud movements and outflows that changed the seafloor morphology and released up to 43000 m3 of methane14. Deep-sea MVs are often associated with the presence of shallow gas hydrates15 and are known to sustain chemosynthesis-based seafloor ecosystems referred to as cold seeps16. However, the current understanding of the mechanisms driving fluid flow, the fluid sources , the discharge volumes of volatiles and the impact of mud volcanism on seafloor ecosystems is poorly constrained . To move forward in the understanding of these interrelated issues requires systematic investigations of submarine MV systems to detect the sites of seepage relative to MV morphologies, quantify the fluid discharge, and characterize the composition of fluids emitted at and in the vicinity of the locations of active mud extrusion.Less is known about the expressions of activity (quiescent or eruptive) of submarine MVs although estimates of their number , the Mediterranean Ridge, the Anaximander Mountains, and the Nile deep sea fan . On the Mediterranean Ridge, scientific drilling of two MVs19 revealed mud breccia discharge processes20 and a fluid source depth of ~3.5–7 km21. In the Anaximander Mountains region, gas release at MVs supports the formation of shallow gas hydrates22, which have been found to be prone to decomposition during eruptive phases of mud volcanism15. Investigations of MVs on the Nile deep sea fan documented mud outflows and fluid seepage at submarine brine pools24. The presence of brine indicated migration from below and through fault-ruptured Messinian evaporite layers24. In the main sediment basins of the eastern Mediterranean, however, Messinian salt deposits are thought to act as seals to fluids ascending from depth. The MV provinces have in turn been found to predominantly occur where salt deposits are absent, thin, or undetected in seismic data18.MVs occur globally in a variety of plate tectonic settings, but are most common along convergent margins within accretionary prismsfan Fig. 18. On th25 . Evidence of active gas emissions into the water column (gas flares) and mud breccia outflows were found only at Venere MV27. A recent study on the extrusive history of Venere MV presented evidence of mud breccia discharge for at least the last 800 years at rates as high as 47000 m3/year, suggesting a long-term pressurized state of the MV system27. These findings contrast with the typical view of short-lived massive mud breccia extrusions interrupting long-term quiescent-type seepage. They require a more detailed study of the present-day activity of Venere MV, including potential fluid sources.At least 54 extrusive features have been documented across the inner regions of the Calabrian accretionary prismin-situ pressure, and fluid analyses. The multi-parameter datasets are used to study processes involved in active mud volcanism. The processes that drive mud volcanism typically occur at depths not readily accessible for direct sampling, thus the results allow for inferences on crustal dewatering and hydrocarbon generation at depth within the CAP. Element and fluid cycling and contribution of elements to the hydrosphere, which may be applicable to other convergent margins are discussed. Finally, the findings of this study support a new conceptual model involving coeval mud breccia extrusion, faulting and gas seepage, in which subsurface fluid migration pathways are linked to different expressions of seafloor fluid discharge and ecosystem distribution.In this study the nature of activity at a submarine MV is investigated, based on a multi-method approach involving hydroacoustic and visual seafloor datasets acquired by AUV (autonomous underwater vehicle) and ROV (remotely operated vehicle) surveys, seafloor sediment samples, gas samples collected under 29. The incorporation of evaporites resulting from the Messinian salinity crisis (late Miocene) into the accretionary prism strongly influenced the structure of the CAP. The CAP can be divided into an external (post-Messinian) wedge that hosts evaporites and an internal (pre-Messinian) wedge hosting mainly clastic sediments affected by out-of-sequence thrusts, the most prominent of which is referred to as the Calabrian Escarpment . The internal wedge is composed of accreted Mesozoic-Cenozoic units overlain by forearc basins (Spartivento-Crotone basins33). Late Pleistocene uplift has resulted in exposure of the inner parts of the forearc basins in southern Calabria, where sediments date back to the middle Miocene  and contain Messinian deposits including <200 m-thick evaporites within the onshore Crotone forearc basin35 and in nearshore wells36. Seismic reflection profiles of the offshore forearc basins indicate up to 2 km of sediment, overlying thrust structures . These sequences are inferred to also date back to the Tortonian and to contain Messinian deposits36. However, the sedimentological composition of these deposits – i.e. whether they contain evaporites – remains unknown.The present-day CAP in the central Mediterranean Sea Fig.  developeCE; Fig. 30–32. Th25. MVs in both tectonic settings are argued to have been erupting since ca. 3.5 Ma (early Pliocene), based on seismic profiles across two structures. These two MVs are associated with buried extrusive edifices over 1 km thick that interfinger with the upper part of the Plio-Quaternary succession2. The geometries of these and other MVs have been argued to reflect the interaction of fluids rising from within the prism with near-surface structures, including thrusts beneath the forearc basins25. Mud breccia from several MVs contained sediment derived from strata ranging in age from Recent to as old as Late Cretaceous, consistent with sources at depth within the accretionary prism39. Most MVs are associated with seafloor backscatter signatures interpreted to indicate mud breccia outflows over the last glacial to interglacial cycle25. Evidence of presently ongoing fluid release on the CAP is limited, however, to reports of sediment cores releasing gas and findings of small tubeworms in a single core40. Previous studies of MVs on the CAP lack a quantification of the gas or characterization of the involved fluids. Cold seep ecosystems represent important ecological hotspots in the Mediterranean Sea but they are still poorly known on the CAP.The majority of the 54 MVs so far identified in the CAP are located across the internal (pre-Messinian) accretionary prism, within the forearc basins and among the thrust-fold belt to seawardThe seafloor morphology of Venere MV is imaged in detail by AUV-borne swath bathymetric data with a horizontal resolution of up to 1.6 m Fig. . BathymeAt the western cone , elongate mudflow deposits are observed extending for horizontal distances of up to ~1,600 m down the southern flank to the foot of the cone .At the eastern cone, bathymetric observations reveal elongate but smooth lobes Fig. , consistHydroacoustic surveys conducted 28 times across Venere MV over a 31 day period during M112 revealed temporally variable but generally persistent gas discharge (flares) from five sites: one slightly below the western summit (Site 3) and four peripheral to the MV cones , heavier hydrocarbons such as ethane (C2) and propane (C3) were detected in gas samples from the mud breccia, with traces of butane (C4) and pentane  in gas samples from the mud breccia ranged between 79 and 107. Methane (CH4) constituted 98.5 vol-% of the total gas released from the pressure core and using an average sediment porosity of 0.5 (determined in samples from gravity core GeoB19245-1), the calculated average CH4 concentration was 321 mmol/L pore water. This CH4 concentration is about 2.7 times higher than the calculated in-situ CH4 solubility  at 2 mbsf = 31.33 °C; salinity (S) = 10 psu; following equations by Tishchenko et al.41). The stable carbon and hydrogen isotopic composition of methane  ranged from −36.6 to −41.6‰ V-PDB and from −143.9 to −156.0‰ V-SMOW, respectively  and propane (C3). Gas bubbles sampled at the peripheral seeps had C1/C2+ ratios of 859–2249, with δ13C-CH4 and δD-CH4 ranging from −44.7 to −48.6‰ V-PDB and from −176.0‰ to −182.6 V-SMOW, respectively . This does not preclude the occurrence of a pressurized reservoir trapping these gases at shallower depths (i.e. cooler temperatures) below Venere MV.Formation temperatures range from ~70 °C for the early thermogenic hydrocarbons to upwards of 150 °C for late thermogenic, methane-rich gas production systemset al.47). An absence of gas hydrates at the western summit of Venere MV is consistent with measured sediment temperatures, which shift mud breccia at >30 cmbsf outside the gas hydrate stability zone , the temperature range of 60–150 °C translates to fluid generation at 3–7.5 km, in accordance with the formation depths required by the thermogenic hydrocarbons.Mud breccia at the western summit of Venere MV is also characterized by pore water freshening Fig. . The mag32). On the CAP, MV sources have previously been inferred to underlie the forearc-basin sediments based on mud breccia compositions containing fossils and clasts derived from strata as old as Cretaceous39. It is unknown if Messinian deposits in the Crotone forearc basins contain evaporites, however, at Venere MV the mud breccia containing strongly freshened pore waters offers no evidence of interactions with Messinian evaporites during their rise from depth. One possibility is that Messinian deposits, if present in the sedimentary succession below the location of Venere MV, did not contain evaporites. This possibility is in accordance with other studies, which found that MVs in the eastern Mediterranean region preferably developed at sites where Messinian deposits are thin or absent36 or cut by deep-seated faults acting as fluid-migration pathways24. Another option is that, over time, fluid migration through the plumbing system beneath Venere MV has removed any Messinian evaporites.These results indicate that formation of thermogenic hydrocarbons and water release by mineral dehydration reactions led to the generation of the fluids that are associated to the mud breccia at Venere MV. These processes require temperatures ≥60 °C that, at the location of Venere MV, indicate source depths exceeding 3 km. These depths lie well below those of the inferred depth range of Messinian deposits interpreted to occur in the Crotone forearc basin Fig. 32. On th49. However, the processes leading to the co-occurrence of quiescent-type activity and active mud breccia extrusions are not well understood. Based on observations of the prevailing seafloor morphologies and processes, and analyses of pore fluid (gas and water) compositions of samples from Venere MV, a conceptual model of its plumbing system is proposed that inter-relates extrusive processes, ring-faulting, fluid seepage and seafloor ecosystems . The resulting estimate of the mud breccia flux through the conduit of 3800 to 38000 m3/year fits well with published rates of 5000 to 47000 m3/year for mud breccia extrusions at Venere MV over the last ~882 years27.The rough surface structure of exposed mud breccia observed at and below the extrusion site Fig.  together27. Considering the timing of mud breccia extrusions from the western cone (recent to on-going) and the quiescent-type gas release along the ring faults (long-term and on-going) it is argued that both processes have been occurring coevally.The deeply-rooting conduit inferred to rise beneath the western summit is encircled by inward-dipping ring-faults. Gas discharge at peripheral sites indicates that fluids are migrating along the faults. The peripheral seeps of Venere MV are so far the only sites on the CAP from which extensive chemosynthesis-based communities associated with gas emissions and authigenic carbonate deposits have been documented. These observations indicate a long-term upward supply of dissolved methane at Venere MV. The presence of decimetre-thick authigenic carbonates in particular indicate persistent gas seepage over timescales of thousands of years27. Faults bordering MV edifices are a known features of submarine MVs . In a deep-marine environment, however, the high-resolution bathymetric dataset presented here reveals in unprecedented detail the seafloor expression of a ring-fault bounded caldera with sites of active fluid release along it. The stable carbon and hydrogen isotopic signatures of methane collected at the peripheral seeps  mud breccia outflows, extrusion of thermogenic hydrocarbons, and freshened pore waters at a summit site; and 2) long term advective seepage of methane fuelling cold-seep communities at sites along peripheral ring-faults. Mud breccia extrusion, gas release along peripheral seeps, and caldera ring-faulting appear to be linked and sustained by persistently high pore fluid pressures. Fluids discharging with mud breccia at the summit are generated by thermogenic organic matter degradation and mineral dehydration reactions occurring at depths of >3 km. This formation depth implies that fluid sources are located below the Messinian to Recent sedimentary infilling of the Crotone forearc basin. Freshened pore waters indicated that Messinian evaporites are either not present or have been removed by fluid migration from below Venere MV.Considering the presence of thermogenic hydrocarbons in both fluid domains, it is inferred that seepage at Venere MV involves an upward-branching plumbing system and that overpressured gas diverted laterally from the main MV conduit migrates upward and mixes with gas of shallow origin. As a result, chemosynthesis-based communities that rely on persistent fluid supply and thick authigenic carbonates are limited to the peripheral seeps along the ring faults of the caldera. An upward-branching plumbing system that experiences high subsurface pore pressures can explain how both mud breccia discharge and quiescent-type seepage co-exist at an individual MV. Such a mechanism may allow for mud breccia discharge to be sustained over timescales exceeding the short-lived eruptions typically associated with active mud volcanism. Furthermore, the upward-branching fluid migration pathways exert an important control on the distribution of seepage-dependent chemosynthesis-based oases of life around submarine MVs.26 and R/V POSEIDON cruise POS49957. Swath bathymetry data were collected using two Kongsberg multibeam systems, a ship-borne EM122 (12 kHz) in 2014 and an EM2040 (300 kHz) carried by the autonomous underwater vehicle (AUV) MARUM-SEAL 5000 in 2014 and 2016. Bathymetric datasets were processed with MB-System58. Ship-based data was gridded to 25 m lateral and meter-scale vertical resolution and AUV-based data to 1.6 m lateral and 0.1 m vertical resolution.Multi-disciplinary investigations of Venere MV were undertaken in 2014 and 2016 during R/V METEOR cruise M112in-situ pressure59. Seafloor observations, sediment sampling with push cores (30 cm long) and collection of gas bubbles from the water column were conducted using the remotely operated vehicle (ROV) MARUM QUEST 4000 m during M11226. In-situ sediment temperatures were measured using miniaturized temperature data loggers , mounted as an array of 6 sensors on a 5 m gravity core barrel or with a 60 cm long probe , inserted vertically into the sediment using the ROV60.Sediment cores up to 5 m long were acquired using a conventional gravity corer and the Dynamic Autoclave Piston Corer (DAPC), the latter allowing for retrieval of cores under 61; from gas bubble sampling under in-situ pressures over the seeps using the ROV  of methane (CH4) were determined by gas chromatography coupled to isotope ratio mass spectrometry (GC-IRMS) at MARUM. Repeated analyses of standards gave a reproducibility for C1/C2+ ratios, stable carbon, and hydrogen isotope ratios of ≤2%, 0.5% and 1%, respectively. Values of ethane (C2) and propane (C3) for GeoB19251-1 were analyzed by GEO-data .Pore fluids were extracted from gravity cores and push cores using rhizon samplers. Sulphate and chloride concentrations were determined by ion-chromatography (“882 Compact IC plus 1” by Metrohm on a “Metrosep A Supp 5” column) with an analytical error below 0.4% for both anions. Hydrocarbon data was generated from sediment by headspace techniqueROV Fig. ; and fro62. Calculations were performed for gas compositions obtained from mud breccia .Relevant data will be made available on PANGAEA® (Dataset 1


HHIP) are robustly associated with chronic obstructive pulmonary disease (COPD). Hhip haploinsufficiency in mice leads to increased susceptibility towards the development of emphysema following exposure to chronic cigarette smoke (CS). To explore the molecular pathways which contribute to increased susceptibility, we performed metabolomic profiling using high performance liquid chromatography tandem mass spectroscopy (LC/MS-MS) on plasma, urine, and lung tissue of Hhip+/− heterozygotes and wild type (Hhip+/+) C57/BL6 mice exposed to either room-air or CS for six months. Univariate comparisons between groups were made with a combined fold change ≥2 and Student’s t-test p-value < 0.05 to denote significance; associations with mean alveolar chord length (MACL), a quantitative measure of emphysema, and gene-by-environment interactions were examined using empiric Bayes-mediated linear models. Decreased urinary excretion of cotinine despite comparable plasma levels was observed in Hhip+/− heterozygotes; a strong gene-by-smoking association was also observed. Correlations between MACL and markers of oxidative stress such as urinary methionine sulfoxide were observed in Hhip+/− but not in Hhip+/+ mice. Metabolite set enrichment analyses suggest reduced antioxidant capacity and alterations in macronutrient metabolism contribute to increased susceptibility to chronic CS-induced oxidative stress in Hhip haploinsufficiency states.Genetic variants annotated to the hedgehog interacting protein ( Recent genome-wide association studies (GWAS) have consistently identified a COPD susceptibility locus in an intergenic region on chromosome 4q312. Using chromosomal conformation capture studies, our group demonstrated that this GWAS region interacts with the hedgehog interacting protein (HHIP) promoter through a functional genetic variant within a distal enhancer which alters binding to the SP3 transcription factor3. Subsequent studies of human lung epithelial cells exposed to cigarette smoke in vitro suggested that, in addition to its established roles in morphogenesis and embryonic development through the hedgehog pathway4, HHIP may alter extracellular matrix and cell growth pathways5. Recently, we also demonstrated a role for HHIP in the development of spontaneous, age-related emphysema in murine models of Hhip haploinsufficiency6.Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the United StatesHHIP and exposure to chronic cigarette smoke (CS), the leading environmental risk factor for COPD, we studied a murine model of HHIP haploinsufficiency generated on a C57/BL6 background. While homozygous Hhip−/− mice die shortly after birth due to defects in lung branching morphogenesis, Hhip+/− heterozygotes are viable, have normal lung development, and exhibit an approximately 33% reduction in the expression of Hhip, a level comparable to that observed in human COPD lung tissue samples harboring HHIP GWAS risk variants3. Hhip+/− heterozygote mice demonstrate an increased susceptibility towards the development of both functional and histological emphysema when exposed to chronic cigarette smoke7; network analysis of lung gene expression data demonstrated an enrichment of lymphocyte activation pathways in Hhip+/− mice relative to similarly exposed wild type mice7. To date, investigations into the metabolic perturbations which may contribute to the increased susceptibility towards the development of emphysema in Hhip+/− heterozygotes have not been performed.To explore the interaction between HHIP haploinsufficiency on the development of COPD, metabolomic profiling using an untargeted liquid chromatography-tandem mass spectroscopy (LC-MS/MS) platform was performed on the plasma, urine, and lung tissue from Hhip+/− heterozygote and Hhip+/+ wild type mice exposed to either room air or 6 months of cigarette smoke .Metabolomic profiling is a relatively novel “-omics” platform where the comprehensive small molecule composition of a biological material is assessed. As such, metabolomics represents a more proximal and integrative snapshot of the environmental and genetic risk factors which likely contribute to a disease phenotype. Unlike the genetic profile which remains largely invariant within a given individual, multiple “metabolomes” representing different biological materials, physiological states, and exposure conditions can exist in a single organism. To further explore the impact of Hhip+/+, CS-exposed Hhip+/+, room air-exposed Hhip+/−, and CS-exposed Hhip+/−. Although profiling was performed using an untargeted platform, because we wished to perform downstream pathway and enrichment analyses, we limited our analyses to identified compounds. In total, 319 compounds were identified in plasma, 197 in urine, and 323 in lung. Fatty acids were not detected in urine samples and account for the decreased number of metabolites reported in urine. The overlap between metabolites in each dataset by sample type are shown qualitatively in Supplementary Figure Five mice were analyzed from each group in this 2 × 2 experimental design: room air-exposed Hhip genotype (Hhip+/− heterozygotes versus Hhip+/+ wild type) under room air conditions are shown in Table Hhip+/− mice relative to Hhip+/+ mice. No metabolite met both the fold change (≥2) and statistical threshold for significance in lung tissue.The spectral peak intensity of each metabolite, which is proportional to concentration, was analyzed following log transformation and Pareto scaling. Metabolites demonstrating a minimum ≥2 fold change and a Students t-test p-value (unadjusted) <0.05 between groups were considered significant. Differences in metabolism by Hhip+/+  conditions in both /+ Table  and Hhipce Table . As a prtes Fig.  and may Hhip+/+ wild type to Hhip+/− heterozygotes exposed chronic CS, a more modest number of metabolites were identified as existing in different concentrations by genotype  in lung upon exposure to chronic CS; these increases are not observed in Hhip+/− heterozygotes; the differences in C30:1 PC and C32:2 PC are significant when comparing Hhip+/+ and Hhip+/− groups that have both been exposed to chronic CS. Additionally, reduced levels of gentisate were observed in the plasma and lung tissue of Hhip+/− exposed to chronic CS.Because of the relative paucity of metabolites which met our stringent criteria for significance in lung tissue in the majority of our analyses, we performed secondary exploratory analyses using a less stringent threshold of 1.5x fold change  to denote significance. Comparisons between To explore gene-by-environment interactions, we constructed empiric Bayes’ mediated linear models with raw metabolite peak intensities as the dependent variable; dichotomized variables for the main effects and a multiplicative interaction term were modeled as the independent variables as shown below:permutation = 0.002). Only fructose/glucose/galactose were identified in both the urine and lung analyses while C58:12 triacylglycerol overlapped between the plasma and lung analyses.Metabolites with an association p-value < 0.05 for the interaction term were considered significant. Metabolites with significant gene-by-environment interactions are listed in Table 8. Empiric Bayes-mediated linear models were constructed for each experimental condition using the raw metabolite peak values and mean MACL as the dependent and independent variables, respectively; metabolites with an association p-value < 0.05 were considered significant.The mean alveolar chord length (MACL), a quantitative histological measurement of the average vertical and horizontal distances between the alveolar walls, was used as a surrogate for emphysema severity and was determined according to previously published protocolsHhip+/+ mice exposed to room air; these associations were no longer present following exposure to chronic CS nor was it present in Hhip+/− heterozygotes under either experimental condition  ≤ 0.05 was considered significant.Metabolites significant in the (a) univariate analyses by both fold change and p-value testing, (b) gene-by-smoking analysis, and (c) MACL analyses above were mapped to the appropriate unique identifier in the Human Metabolome Database (HMDB ID); these identifiers were used as input for MSEA and pathway analyses as implemented in MetaboAnalyst 3.0Hhip+/− heterozygotes mice exposed to chronic CS versus room air were enriched for several processes. An enrichment of plasma metabolites annotated to RNA transcription was observed, while metabolites annotated to the urea cycle, ammonia recycling, and the citric acid cycle were enriched in the urine .When we performed a similar analysis on metabolites identified in the gene-by-smoking analysis, a trend towards enrichment in metabolites annotated to protein synthesis (FDR = 0.07) was identified in the analysis of urine. As a subgroup analysis, we performed pathway enrichment analysis using the 4 overlapping metabolites identified in both plasma and urine gene-by-environment analyses and noted a significant enrichment of metabolites annotated to (1) glyoxylate and dicarboxylate metabolism and (2) the citrate cycle pathways  is a highly conserved, vertebrate-specific protein which is both induced by and serves as a negative regulator of hedgehog signaling11. HHIP has an established role in branching morphogenesis of the lung during embryonic development4; based on subsequent RNA interference studies in a human airway epithelial cell line, HHIP was also implicated in lung extracellular matrix and cell growth pathways5. In addition to associations with adult height12 and several malignancies13, variants annotated to the HHIP locus have been robustly associated with lung function and the development of COPD14. To date, the mechanisms which underlie the association between HHIP and late-onset (adult) complex diseases have not been fully characterized. A murine model of Hhip haploinsufficiency which closely mimics the impact of genetic variants identified by genome-wide association studies on the gene expression of HHIP in the lung demonstrates increased susceptibility towards the development of emphysema upon exposure to chronic cigarette smoke7; using this model, we explored both baseline differences in metabolism as well as differences induced by exposure to a significant environmental challenge .The hedgehog family of proteins is classified as growth factors and morphogens which mediate an expansive number of processes during embryogenesis and developmentHhip function results in perinatal lethality, haploinsufficiency at the Hhip locus does not appear to significantly alter the viability or lung morphology relative to wild-type mice7 under normal conditions. Likewise, baseline differences in metabolism between Hhip+/− and Hhip+/+ mice also appear to be modest. Decreased levels of C6 and C8 carnitine were detected in the plasma of Hhip+/− heterozygotes; both compounds belong to the broader category of acyl carnitines, a group of metabolites involved in fatty acid oxidation. Interestingly, increased plasma levels of C6 and C8 carnitine are characteristic of medium chain acyl-CoA dehydrogenase deficiency , an autosomal recessive disorder characterized by an intolerance to fasting16. Although the functional impact of decreased levels of plasma C6 and C8 carnitine is not known, decreased levels of L-carnitine in lung tissues were recently reported in a porcine pancreatic elastase murine model of emphysema17.While complete loss of Hhip+/+ wild type and Hhip+/− heterozygotes. Decreased urinary excretion of pantothenate, an essential nutrient also known as vitamin B5 which is involved in the synthesis of coenzyme A (CoA) and the metabolism of almost all macronutrients , was observed in both Hhip+/+ and Hhip+/− mice exposed to cigarette smoke. In both instances, a corresponding increase in plasma levels was not observed. Thus, whether cigarette smoke exposure alters the synthesis or absorption of the nutrient from the gastrointestinal tract or the localization, turnover, or sequestration of pantothenate in different tissues remains unknown. Interestingly, increased levels of pantothenate have been reported in a human alveolar lung tissue cell line exposed to mainstream cigarette smoke in vitro18; this was not observed in our data which was ascertained on whole lung tissue samples from an in vivo exposure model.Exposure to cigarette smoke appears to elicit a number of changes to metabolite concentrations in the plasma and urine of both Hhip+/+ and Hhip+/− mice exposed to chronic CS was guanine, a purine derivative which is one of five bases integral to DNA and RNA. Increased urinary guanine in CS-exposed mice was one of the strongest associations in our study, remaining robust even after application of a conservative Bonferroni adjustment for multiple testing. Interestingly, several derivatives of guanine, such as 8-hydroxydeoxyguanosine and 7-methylguanine, have been associated with cigarette smoking and may reflect levels of oxidative damage and DNA methylation, respectively21; these guanine derivatives were not assessed directly in our study and represent potential avenues for future investigations.Another metabolite consistently altered in both 22 and was significantly increased in the urine and plasma of CS-exposed Hhip+/+ and Hhip+/− mice. The finding of increased levels of cotinine and a relative depletion of glutathione in the plasma of Hhip+/+ wild type mice exposed to chronic cigarette smoke is also consistent with known hepatic xenobiotic detoxification pathways23 and serves as a proof-of-concept finding. Interestingly, when we compared Hhip+/+ and Hhip+/− mice which had both been exposed to chronic CS, Hhip+/− heterozygotes had significantly lower urinary cotinine relative to Hhip+/+ wild type mice. The decreased urinary excretion of cotinine in Hhip+/− heterozygotes occurs despite similar plasma cotinine levels in both strains and does not appear to be due to differential renal function as assessed by plasma creatinine levels . Previous studies have demonstrated a strong correlation between plasma and urinary levels of cotinine in both humans and mice 23; a strong inverse correlation between plasma and urinary cotinine levels was noted for Hhip+/+ wild type mice exposed to cigarette smoke , but no correlation was noted in Hhip+/− heterozygotes   Fig. . WhetherHhip+/+ and Hhip+/− mice exposed to chronic CS relative to mice exposed to room air, however no difference in cotinine excretion was found when we compared Hhip+/+ and Hhip+/− mice who had both been exposed to chronic CS. However, due to limited sample availability, urinary creatinine levels could not be assayed, thus these values were not normalized to urinary creatinine excretion. Interestingly, while a trend towards increased plasma cotinine was observed in both Hhip+/+ and Hhip+/− mice exposed to chronic CS, these differences did not reach statistical significance in either group despite known and controlled exposure to cigarette smoke. Whether this is due to the rapid clearance of cigarette smoke metabolites in plasma or due to limitations of the assay employed remains undetermined. We similarly support the mass spectroscopy-based findings of reduced in urinary creatine observed in Hhip+/+ mice exposed to chronic CS as well as the lack of decrease in urinary creatine excretion in Hhip+/− heterozygotes. There was, again, no statistically significant difference by genotype when comparing the two groups of mice exposed to chronic CS, but these values were similarly not adjusted for urinary creatinine excretion.We were able to support some of the mass spectroscopy-based results reported above using ELISA and colorimetric assays in a largely non-overlapping cohort of mice from the same experimental conditions. Urinary cotinine excretion was increased among both Hhip+/− heterozygotes towards developing more severe histological and functional emphysema when exposed to chronic CS or during aging relative to their wild type counterparts are incompletely understood but are likely mediated through an increased sensitivity towards oxidative stress6. Given the “standardized” exposure to oxidative stress in our experiment, we hypothesize that this increased sensitivity may be due to a) reduced antioxidant capacity and b) changes in metabolism which perpetuate oxidative damage. Findings which support the hypothesis of reduced antioxidant capacity in Hhip+/− heterozygotes include reduced levels of gentisate in plasma and lung ; future studies involving a larger number of observations will allow for more detailed analyses of more complex interactions. Lastly, we acknowledge that, because our model is murine and examined only female mice, the generalizability of our findings to both genders and extrapolation to human metabolism is not known; thus, future metabolomic investigations in both genders and using human populations are warranted.Our exploratory analyses using metabolomic profiling of a murine model of Hhip+/− mice and the chronic cigarette smoke exposure conditions have been previously published7. All protocols were in compliance with NIH recommendations for the Care and Use of Laboratory Animals; all protocols were approved by the Harvard Medical Area Standing Committee on Animals (Protocol #: 04833). Female Hhip+/− heterozygotes on a C57/BL6 background and their female wild type littermates were subjected to either mixed main-stream and side-stream cigarette smoke (3R4F Kentucky Research cigarettes) for 5 days per week or filtered room air starting at age 10 weeks for a total duration of 6 months.Details regarding the generation of Hhip+/+, CS-exposed Hhip+/+, room air-exposed Hhip+/−, and CS-exposed Hhip+/−) were subjected to metabolite profiling. Urine was collected immediately prior to euthanization by placing each mouse on a clean surface and aspirating spontaneously voided urine using a sterile pipette. Urine was transferred into a sterile 1.5 mL centrifugation tube and was immediately stored at −80 °C until analysis. A new, clean surface was used for each mouse. Euthanization by CO2 narcosis and cervical dislocation was then performed and 500 μL of blood was collected from the right ventricle immediately using a 26-gauge needle. Blood was transferred into 1.5 mL tubes coated with heparin and centrifuged  to separate plasma. Plasma aliquots were transferred into new 1.5 tubes and stored immediately at −80 °C until analysis.Plasma, urine, and lung tissue from five mice from each experimental condition  and were snap-frozen for metabolomic profiling. Lung tissue was homogenized in 6 volumes of water using a bead mill  and the aqueous homogenate (30 μL) was subjected to protein precipitation using four volumes of 80% methanol containing inosine-15N4, thymine-d4, and glycocholate-d4 internal standards . Samples were then centrifuged  and aliquots of the supernatant (10 μL each) were subjected to LC-MS profiling methods (below).Murine lungs were harvested after blood collection for mean alveolar chord length (MACL) measurements as described previously36; additional details are also provided in the Online Supplement – Methods section. MultiQuant software (v1.2) was used for automated peak integration; all peaks were manually reviewed and compound identities were confirmed using reference standards and reference samples37. Lung tissue profiles were adjusted for tissue dry weight and urinary profiles were normalized by creatinine levels (assayed as one of the metabolites included in our mass spectroscopy-based platform).Untargeted metabolite profiling was performed using liquid chromatography tandem mass spectroscopy (LC/MS-MS) as previously described9 and R . Samples with >40% missingness and metabolites with >30% missingness were removed; remaining missing values were replaced with a small value . Data were log transformed and Pareto scaled prior to conducting univariate analysis by genotype (Hhip+/+ versus Hhip+/−) or smoke exposure (room air versus CS). Metabolites which demonstrated a minimum fold change of 2 and a Students t-test p-value < 0.05 were considered significant. Metabolites that additionally met statistical significance following correction for multiple testing using an false-discovery rate (FDR) < 0.05 were denoted in the results table. Gene-by-smoking interactions and associations between mean alveolar chord length and metabolites were tested using empiric Bayes-mediated linear models as implemented in the limma38 package for R. An association p-value < 0.05 was considered significant.Data cleaning and formatting were performed in MetaboAnalyst 3.09. Over-representation analysis (ORA) was performed using the hypergeometric test using metabolite sets based on normal metabolic pathways and the reference pathway library for Mus musculus available through the Kyoto Encyclopedia of Genes and Genomes (KEGG)39, respectively. An FDR ≤ 0.05 was considered significant.Metabolite set enrichment analysis (MSEA) and pathway analyses were performed using MetaboAnalyst 3.0Hhip+/+ and Hhip+/− mice using the Calbiotech Mouse/Rat Cotinine enzyme-linked immunosorbent assays (ELISA) kit . Quantitative measurements of urine creatine were made using the Abcam colorimetric/fluorometric creatine assay kit  in Hhip+/+ and Hhip+/− mice exposed to chronic cigarette smoke. Standard curves were generated according to the manufacturer’s guidelines for each assay. Comparisons between groups were made using a Student’s t-test with a p-value < 0.05 to denote significance.Validation of several metabolites was performed on plasma and urine samples collected from a partially overlapping group of mice (n = 20). Cotinine concentrations were determined in the plasma and urine of Online Supplement