A uraninite fission zone comparable to the Oklo reactors in Gabon can produce 0.1‒1 mol m−2 year−1, orders of magnitude greater than other scenarios of FA production or delivery for which reaching sizeable concentrations of FA are problematic. Radioactive mineral deposits may be favorable settings for prebiotic compound formation through emergent geologic processes and FA-mediated organic chemistry.Water creates special problems for prebiotic chemistry, as it is thermodynamically favorable for amide and phosphodiester bonds to hydrolyze. The availability of alternative solvents with more favorable properties for the formation of prebiotic molecules on the early Earth may have helped bypass this so-called “water paradox”. Formamide (FA) is one such solvent, and can serve as a nucleobase precursor, but it is difficult to envision how FA could have been generated in large quantities or accumulated in terrestrial surface environments. We report here the conversion of aqueous acetonitrile (ACN) via hydrogen cyanide (HCN) as an intermediate into FA by γ-irradiation under conditions mimicking exposure to radioactive minerals. We estimate that a radioactive placer deposit could produce 0.1‒0.8 mol FA km Laboratory studies, however, have revealed several problems with the abiotic synthesis of these polymers prior to life’s emergence. Specifically, though the prebiotic synthesis of RNA monomers has recently been reported from relatively simple molecules5, only a few geochemical scenarios that can facilitate these synthesis reactions have been experimentally investigated7. A frequent criticism of many model prebiotic reactions is that it is unlikely the needed precursor ingredients would ever spontaneously reach the high concentrations typically employed, giving rise to a puzzle known as the Concentration Problem8. But perhaps the most deleterious barrier to the emergence of life arises from the most critical ingredient of life itself ‒ water9. The average half-life for hydrolysis of a peptide bond is on the order of 102 years at pH 7 and 25 °C10, and the half-life of an RNA phosphodiester bond at 30 °C is estimated to be less than one year under the same conditions11. Water therefore hydrolyzes biopolymers such as nucleic acids and proteins over relatively short time-scales, but is also a necessary solvent medium for all known life. This fundamental mismatch between initial and final chemical constraints on life’s origins leads to what is known as the Water Paradox12. The Concentration Problem and the Water Paradox present formidable conceptual problems for any origins of life theory, including “RNA World” scenarios that necessitate abundant, abiotic production of polynucleotides2.Abiotically produced nucleic and amino acid polymers are widely viewed as key chemical intermediates that link a lifeless Earth with the universal ancestor of all known life2, FA), which is a liquid under normal terrestrial surface temperature and pressure conditions, has been advanced as an alternative solvent to water that could enable chemical complexification and a means of bypassing the Water Paradox15. FA is a polar solvent that has the advantageous properties of promoting dehydration condensation reactions17, solubilizing phosphate minerals18, and serving as a feedstock for several biologically relevant compounds including nucleobases21, amino acids and carboxylic acids when heated and in contact with a variety of mineral catalysts25. FA can be produced via the hydration of hydrogen cyanide (HCN) or the decomposition of ammonium formate, but the competing reaction of FA hydrolysis to formic acid constrains maximum possible aqueous concentrations to approximately 10−5 M28. These dilute amounts are insufficient to validate the use of neat concentrations of FA employed in some model prebiotic syntheses, or to serve as a reservoir for abundant nucleobase production32. Other direct sources of FA that bypass bodies of water such as cometary33 or meteoritic34 delivery have been invoked in conjunction with ‘desert-like’ periodically dry settings or hydrothermally-driven thermophoresis as a means of concentrating FA35, but such scenarios necessitate ideal and perhaps implausible circumstances to simultaneously produce and then concentrate FA to any appreciable reservoir size36.Formamide  may reduce problems associated with reaching high concentrations of FA while simultaneously mitigating the Water Paradox. Radiolysis of water produces hydroxyl radicals that increase rates of hydration and hydrolysis above those associated with thermal activation alonee40 Fig. . The enee40 Fig. . Under e−1. GC-MS was used to analyze the resulting mixture. FA was observed along with acetaldehyde, acetamide, acetic acid and succinonitrile as major products  for FA formation from ACN of 0.012 molecules/100 eV. Although the radiolytic yield for FA decreases with lower starting concentrations of ACN, normalizing the G-values to initial ACN molarity reveals that the production of FA becomes more efficient at lower initial ACN concentrations , total dose (over the range of 72 to 3200 kGy) and initial concentration of ACN (0.5‒50 volume %). At 3200 kGy total dose, additional compounds including N-methyl-acetamide, N-methyl-formamide and pyrimidine-like compounds were produced at levels that could not be detected at lower total doses (Table SI-1). Generation of FA seems robust to oxidizing conditions; replacing N2 with a standard atmosphere or adding oxygen-rich nitrate had only a minor effect on FA production.The irradiation of ACN was initially investigated as a 30 volume % (~5.7 M) aqueous solution, which was exposed to an 800 kGy dose at 3kGy hcts Fig. . Hydrogects Fig. , SI-1. Tcts Fig. . The samons Fig. . Overall3 radicals by homolytic cleavage48.Given the observations described above, we infer a pathway of FA production that proceeds though HCN as an intermediate. The radiolytic generation of HCN from pure ACN has been previously reported, the mechanism of which is proposed to proceed by direct photolysis of ACN generating •CN and •CH49. Addition of a hydroxyl radical followed by rapid tautomeric rearrangement leads to the FA radical.−1 over a range of dose rates revealed FA as a major product detectable by GC-MS (G = 0.08 molecules/100 eV), corroborating the proposed mechanism of FA formation through an HCN intermediate.Following the production of HCN, the generation of FA follows a well-understood mechanism2 and CH4 yield HCN and ACN as two major products51; though HCN appears to be the crucial intermediate to FA production, it is also a more volatile and readily reactive compound, which poses a challenge to having it accumulate to significant concentrations. ACN, on the other hand, is much less volatile, affording it the potential to settle from the atmosphere and to collect in surface water reservoirs along with other, less abundant nitrile species52. Nitriles produced by gamma radiolysis of atmospheric N2 and CH4 proximal to radioactive minerals could have been supplemented by more distributed energy sources such as solar UV, galactic cosmic rays (GCRs) or coronal mass ejections associated with solar flare events53 throughout the atmospheric column, which would have been introduced into the system during periodic influx of external water. The result would have been robust, long-term production of FA in a fixed setting 55. Temperature fluctuations caused by water- or organic-moderated fission of uranium-bearing minerals would periodically raise temperatures above 100 °C, while also permitting influx of new sources of dissolved HCN or ACN from external sources that could be radiolyzed to produce FA. To determine if the concentration of FA is possible in the presence of the other radiolysis products observed . A fissioning mineral seam of uranium, with dimensions comparable to a typical fission zone described from the Paleoproterozoic Oklo deposit, has a combined (radiolysis and heat) power output of about 10 kilowatts58. About 13% of this power is composed of γ or β rays that can penetrate substantial distances beyond their host minerals59. These parameters yield an estimated FA production rate of 0.1–1 moles FA m−2 yr−1, which is over 6 orders of magnitude larger than the estimated FA production from placer minerals or delivery from cometary material per unit area34. The highest area-normalized production rate of about 1 mole FA m−2 yr−1 would require conversion of approximately 10% per day of the HCN present within the penetration range of the mineral seam’s γ radiation, and with HCN at the highest estimated concentration of oceanic reservoirs; a smaller conversion fraction per day would be required for shallow lake reservoirs with higher HCN concentrations26. This estimate does not include direct HCN or ACN production from irradiation of atmospheric gases by the deposit itself, nor does it include diffusion of HCN or ACN from adjacent volumes each day. Within this envelope of parameters, the theoretical upper limit of FA production in shallow terrestrial settings would be limited by efficiency of radiolytic conversion from HCN and ACN feedstocks rather than by the amount of nitrile feedstock abundance. An individual fission zone comparable to those at Oklo could produce an upper limit of 230,000 kg of FA over an average fission zone lifespan of about 500,000 years and within a radius of about 10 meters.The radiolysis yields determined herein enable estimation of the geochemical production parameters of FA within a radioactive mineral deposit. The i.e., FA degradation in water at 30 °C and pH 726) then each maximum area-normalized reservoir is calculated as the ratio of normalized FA input to the degradation rate , rutile (TiO2), pyrite (FeS2) and apatite (Ca10(PO4)6(OH)2)40, some of which promote condensation of FA into nucleobases14. We observed herein that radiolysis of aqueous ACN produces significant amounts of four different amide solvents , all of which are known to support the phosphorylation of nucleosides and nucleotides with solid phase apatite60, yielding multiple pathways and abundant sources of two of three nucleic acid monomer components: nucleobases and phosphate.Radioactive mineral deposits capable of producing abundant FA would place this compound in contact with placer minerals relevant to prebiotic chemistry. Common placer sediments include monazite PORadioactive mineral deposits are unique geochemical environments that can produce FA far more quickly than it is degraded. FA is a demonstrated nucleobase precursor and water-alternative solvent with favorable properties for phosphorylation and polymerization. In conjunction with localized production of abundant FA, these geochemical settings may offer one possible means of mitigating both the Water Paradox and the Concentration Problem, at least with respect to the production of nucleotide components. Periodic drying of placer deposits has the potential to concentrate FA, other high boiling-point solvents, and solutes to a degree which renders condensation reactions favorable. Radioactive mineral deposits open the intriguing possibility that water-alternative solvents could have assisted or even enabled the emergence of polymeric chemical systems that preceded water-based life as we know it.2 three times. For HCN experiments, the solutions were prepared from NaCN and water which had been degassed with N2. The pH of the NaCN solutions were adjusted to 7 using concentrated HCl, after which the reaction vials were sealed and the solutions were subjected to further purging with N2 for a period of about five minutes to remove any residual O2. For each experiment, two sets of vials were created. One set was exposed to varying doses and dose rates of γ-radiation from a 60Co source  at the Tokyo Institute of Technology’s 60Co Gamma Radiation Facility. These dose rates are roughly 75 times greater than that estimated from natural fission cores46. Another set of vials, which served as controls were kept in a darkened box at room temperature.Reagents  were purchased from Sigma-Aldrich, Japan and were of 99% purity or higher and used without further purification. Purified 18 MΩ water produced by EMD Millipore’s Milli-Q® water purification system was loaded into glass vials which were pre-ashed at 500 °C for three hours to remove organic contaminants. In the case of ACN, the vials were frozen, vacuum purged and back filled with Nm/z 20–500 Da. Chromatography was conducted using an Rtx-WAX® column ; helium was used as the carrier gas at a flow rate of 40 cm sec−1. The oven temperature program was: 2 min hold at 70 °C, from 70 to 240 °C at 10 °C min−1 and then the temperature was maintained at 240 °C for 5 min. Compounds were identified by comparison of their electron impact fragmentation spectra with standards from the NIST standard reference library, and by verification of spectra with off-the-shelf standard reagents. Concentrations were determined by establishing a calibration curve determined from known concentrations of authentic standards. FA heating and concentration experiment mixtures were analyzed in 10% D2O/90% H2O using a Bruker Avance III nuclear magnetic resonance (NMR) spectrometer tuned to measure 13C spectra at 100 MHz and 303 K.Volatiles in radiolysis samples were analyzed with a Shimadzu GCMS-QP2010 Ultra gas chromatrograph-mass spectrometer (GC-MS). Direct injection of liquid samples and head-space sampling were carried out using a PAL (prep and load solution) RTC autosampler. Data processing was conducted using the LabSolution_GCMSsolution version 4.11 software package. The injection method used an inlet temperature of 250 °C and a source temperature of 200 °C. The scan range was set to The datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary Information


The contribution of the diaphragm to inspiration, as assessed by the gastric pressure to transdiaphragmatic pressure ratio, was 31 ± 17% during “vol,” 22 ± 16% during “single-breath” (p = 0.13), and 19 ± 9% during “continuous” (p = 0.0015 vs. “vol”). This study shows that the relationship between the amplitude of the transdiaphragmatic pressure produced by a diaphragm twitch and its counterpart produced by the underlying diaphragm contraction is not unequivocal. If twitch interpolation is interpreted as reflecting motoneuron recruitment, this study supports motor reorganization compatible with “diaphragm sparing” when an inspiratory threshold load becomes sustained.In humans, inspiratory constraints engage cortical networks involving the supplementary motor area. Functional magnetic resonance imaging (fMRI) shows that the spread and intensity of the corresponding respiratory-related cortical activation dramatically decrease when a discrete load becomes sustained. This has been interpreted as reflecting motor cortical reorganization and automatisation, but could proceed from sensory and/or affective habituation. To corroborate the existence of motor reorganization between single-breath and sustained inspiratory loading (namely changes in motor neurones recruitment), we conducted a diaphragm twitch interpolation study based on the hypothesis that motor reorganization should result in changes in the twitch interpolation slope. Fourteen healthy subjects (age: 21–40 years) were studied. Bilateral phrenic stimulation was delivered at rest, upon prepared and targeted voluntary inspiratory efforts (“vol”), upon unprepared inspiratory efforts against a single-breath inspiratory threshold load (“single-breath”), and upon sustained inspiratory efforts against the same type of load (“continuous”). The slope of the relationship between diaphragm twitch transdiaphragmatic pressure and the underlying transdiaphragmatic pressure was −1.1 ± 0.2 during “vol,” −1.5 ± 0.7 during “single-breath,” and −0.6 ± 0.4 during “continuous” (all slopes expressed in percent of baseline.percent of baseline In humans, the net neural drive that is conveyed from the central nervous system to the respiratory muscles results from the integration of multiple inputs by respiratory spinal motoneurons  participated in the study after ethical clearance from the appropriate external authority . All subjects received detailed information regarding the purpose of the study and methods used, and gave their written consent to participate.Fourteen healthy subjects  of which the inspiratory limb could be silently occluded by an inflatable balloon .Surface recordings of the left and right costal diaphragmatic electromyographic activities were obtained using two pairs of skin-taped silver cup electrodes placed in the 7th right and left intercostal spaces, according to the technique described by Verin et al. . These e2O; Validyne, Northridge, CA, USA). Transdiaphragmatic pressure (Pdi) was obtained on-line by deriving Pes and Pga from a third transducer. The pressure signals were sampled at 400 Hz and digitized. The Pdi trace could be displayed to the subjects.Oesophageal and gastric pressures (Pes and Pga) were measured according to standard practice .Bilateral phrenic stimulation was delivered by cervical magnetic stimulation (CMS), using a Magstim 200 stimulator  equipped with a 90 mm circular coil  placed over the 7th cervical spinal process  were measured according to standard practice . The subjects were asked to produce voluntary inspirations from their functional residual capacity (FRC) against an occluded airway  ranging from 5 to a maximum of 45 cm HData were analyzed offline using Chart software . Diaphragm twitches were considered suitable for analysis only if 1) they were obtained at relaxed FRC as determined from the Pes and abdominal displacement traces immediately preceding the stimulation (relaxed twitches) or the beginning of the corresponding inspiratory effort (superimposed twitches); (2) they were associated with an increase in abdominal circumference; and (3) they corresponded to an electromyographic M-wave amplitude differing by less than 15% from the maximal amplitude identified by the recruitment curves , probably in line with changes in abdominal geometry and compliance between the relaxed and the active condition. Such changes are liable to modify the pressure generated by a diaphragm contraction of identical strength  was greater than the baseline Pdi,tw value obtained from the relaxed diaphragm and was generally obtained with a slight underlying inspiratory effort .After verifying normality and homoscedasticity, the results were expressed as mean ± SD, and comparisons were performed using analysis of variance for repeated measures with the Static Pi,max, Pdi,max, and baseline Pdi,tw were within the normal range in all subjects Table .−1 and its Y-intercept “b” was 100 ± 12%, not significantly different from 100% (p = 0.42). The extrapolation of Equation 1 through the abscissa yielded a “predicted” Pdi,max  that was not significantly different from the measured Pdi,max (p = 0.89).Over the range of voluntary contraction force studied, Pdi,tw decreased linearly with the intensity of the underlying volitional inspiratory efforts Figure  accordinThe overall Pga/Pdi ratio, corresponding to the underlying inspiratory effort in the 14 subjects for all levels of volitional effort, was 31 ± 17 Table . Over thFor technical reasons, the results of this procedure were available for only 12 subjects. The amplitude of the superimposed twitches also decreased linearly Figure  with the−1, significantly different from the slope “a” of Equation 1 (p = 0.04). The Y-intercept of Equation 2 was 109 ± 13%, not significantly different from 100% (p = 0.14). In view of the steeper slope of the relationship, the extrapolation of Equation 2 through the abscissa yielded a Pdi,max value that was significantly lower than the measured Pdi,max . In other words, the recruitment of phrenic motoneurons required to produce a given Pdi was greater during the “single breath” procedure than during the voluntary procedure . Over the range of Pdi,single-breath studied, the Pga/Pdi ratio did not vary in 7 of the 12 subjects, and increased slightly but significantly in the remaining 5 subjects. Overall, no systematic difference was observed between “vol” and “single-breath” conditions in terms of the contribution of the diaphragm to the effort underlying . In other words, the recruitment of phrenic motoneurons required to produce a given Pdi was lower during the “continuous” procedure than during the “single breath” procedure.For technical reasons, the results of this procedure were available for only 8 subjects Figure . In two p = 0.015) . If twitch interpolation is interpreted as reflecting motoneuron recruitment ,” Paris, France a “Contrat de recherche triennal Legs Poix de la Chancellerie de l'Université de Paris,” Paris, France, the Fondation pour l'Avenir (AP-RMA-2015-037) and by the “Investissement, d'Avenir ANR-10-AIHU-06” programme of the French Government. Paris, France. Mathieu Raux was supported in part by a scholarship from the “Comité National Contre les Maladies Respiratoires,” Paris, France and in part by a scholarship from the “Fédération ANTADIR,” Paris, France.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. The reviewer ARC and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


In this study we isolated cardiac myofibrils from 3 TTNtv mutants, and 3 with contractile protein mutations  and measured their contractility and passive stiffness in comparison with donor heart muscle as a control. We found that the three contractile protein mutations but not the TTNtv mutations had faster relaxation kinetics. Passive stiffness was reduced about 38% in all the DCM mutant samples. However, there was no change in maximum force or the titin N2BA/N2B isoform ratio and there was no titin haploinsufficiency. The decrease in myofibril passive stiffness was a common feature in all hearts with DCM-associated mutations and may be causative of DCM.Dilated cardiomyopathy (DCM) is an important cause of heart failure. Single gene mutations in at least 50 genes have been proposed to account for 25–50% of DCM cases and up to 25% of inherited DCM has been attributed to truncating mutations in the sarcomeric structural protein titin (TTNtv). Whilst the primary molecular mechanism of some DCM-associated mutations in the contractile apparatus has been studied  Dilated cardiomyopathy is inherited in about 25–50% of cases and more than 50 single genes are linked to inherited DCM, including genes encoding cytoskeletal, Z-disc, nucleo-cytoskeletal, mitochondrial, and calcium handling proteins making DCM a genetically heterogeneous disease. However “pure” inherited DCM, not associated with other symptoms, such as conduction disease, is most often caused by mutations in sarcomeric proteins. Recently titin truncating (TTNtv) mutations have been found in 25% of putative familial DCM (fDCM) cases2 and a disease causing role of these mutations in A band and Z-disk titin has been asserted3. Despite the emerging importance of TTNtv mutations as a cause of familial DCM (fDCM) very little is known about the functional effects of the mutations in human heart muscle.Historically, the prevalence of DCM was considered to be in the range of 1 in 2500–3000, however, newer modalities and heightened diagnostic awareness now estimate the prevalence of DCM of up to 1 in 5004. The high frequency of potentially disease-causing mutations in end-stage DCM patients confirms the finding of Roberts et al., who studied a large population and demonstrated a >96% probability of pathogenicity in end-stage DCM patients with TTN truncation mutations3. In this study we have directly investigated TTNtv heterozygous mutations in human heart tissue samples in comparison with donor heart and DCM-causing mutations in other sarcomeric proteins. Several human samples were analysed on total titin expression level, N2BA/N2B isoform expression ratio, as well as titin phosphorylation level. To determine the mechanism of diastolic dysfunction, the maximum tension amplitude, length dependence of maximum force, and the rates of tension development and relaxation were measured in DCM samples carrying mutations in TTN (p. R23464T fs*41 and p. Y18923*), TNNI3 (K36Q), TNNC1 (G159D) and MYH7 (E1426K).In a previous study of human heart tissue samples we identified 26 potentially disease-causing mutations in 14 genes, 5 of which were in contractile proteins, 4 were in OBSCN and 6 were truncating mutations in the titin gene (TTNtv)TTN gene predicted to cause chain termination that are potentially causative of DCM4. The presence of the variants was confirmed by direct sequencing  were studied in comparison with myofibrils from donor heart and left ventricular muscle samples with mutations in contractile proteins of the sarcomere: troponin C (TnC) G159D, troponin I (TnI) K36Q and myosin heavy chain E1426K . These are termed familial DCM (fDCM) since linkage has been proven with these mutations9. In comparison, all the familial DCM muscle samples with contractile protein or TTNtv mutations produced the same maximum force at all three sarcomere lengths tested11, although there are some cases where force increases whilst Ca2+-sensitivity does not12.Maximum isometric force depended on sarcomere length, being about maximal at 2.2 µm. The mean maximum force in the donor heart muscle myofibrils was 112 ± 7 kPa (n = 18), reduced to 50 ± 4 kPa (n = 16) at 1.8 µm sarcomere length Fig. . These vACT and kTR) nor relaxation kinetics  were dependent on sarcomere length at maximally activating Ca2+ concentrations  with the donor myofibrils, a value compatible with previous measurements, and was not significantly different in any of the DCM mutant heart myofibrils . kTR, which measures the rate of active crossbridge attachment, was 0.64 ± 0.03 s−1 (n = 25) for the donor heart myofibrils and very similar for the DCM mutant myofibrils (mean kTR = 0.68 ± 0.04); there was a trend to a slightly higher kTR in the mutant myofibrils that was statistically significant for D9 (1.16-fold higher) and D15 (1.18-fold higher). kTR was consistently lower than kACT, especially during the first contraction-relaxation cycle, however the difference was diminished with every new cycle. Fmax, after quick release-restretch, also did not fully recover. This shows that the state was progressively altered. Stretching may depress myofibril activation kinetics and force development in human cardiac myofibrils, but it is also possible that this effect could be related to a deterioration in the myofibril samples , corresponding to a Young’s modulus of 170 kPa at 2.2 µm sarcomere length  in the donor heart samples and was not significantly different in any of the mutant samples. The increase in passive tension with length was less than donor for all of the mutant samples. For the donor heart samples, the parameter gth Fig. . We foungth Fig. .2+-sensitivity by PKA-mediated phosphorylation of TnI at Ser 22 and 23 and it has been proposed that this uncoupling is sufficient to induce the DCM phenotype14. We therefore investigated the Ca2+-regulatory properties of phosphorylated and unphosphorylated troponin isolated from ventricular or atrial muscle using the in vitro motility assay. Donor heart troponin showed the normal modulation of Ca2+-sensitivity. Troponin from the heart samples with mutations in contractile proteins was uncoupled; this has been previously shown for TNNI3 K36Q and TNNC1 G159D in vitro16, but uncoupling due to a myosin heavy chain mutation (MYH7 E1426K) is novel. Three TTNtv heart samples showed normal modulation of Ca2+-sensitivity by TnI phosphorylation, while D6 and D7 showed a blunted relationship of TnI phosphorylation to Ca2+-sensitivity . In contrast to the studies on human heart tissue from HCM patients24.It is also apparent that the contractile protein fDCM and TTNtv mutant phenotypes are distinct from idiopathic DCM or HFpEF since neither N2BA/N2B ratios or contractile protein and titin phosphorylation levels are different from donor heart25, none of which were observed in our adult myofibril samples. The different abnormalities in TTNtv hiPSC-CM and adult myofibrils is most likely due to the immaturity of iPSC-derived cardiomyocytes which functionally resemble foetal muscles26. In contrast, the properties of muscle from transgenic mice with a heterozygous TTNtv mutation27 and our human heart samples are comparable, suggesting that human iPSC-derived cardiomyocytes may indicate congenital effects of TTNtv mutations but do not reproduce the disease-causing process of DCM due to TTNtv observed in adult patients.It is important to note that the changes in contractile properties of adult human tissue samples with TTNtv mutations are very different from TTNtv mutations incorporated into iPSC-derived cardiomyocytes which exhibit sharply reduced contractility, haploinsufficiency, expression of truncated peptides and decreased sarcomere length2+-sensitivity, since kREL increases with increasing EC5011. This was not observed for the TTNtv mutations. In addition, uncoupling provides another potential causative mechanism for contractile protein mutations28. Several recent studies have demonstrated that mutations in thin filament proteins lead to uncoupling of TnI phosphorylation by protein kinase A from the change in Ca2+-sensitivity measured in isolated thin filaments and myofibrils. This leads to a blunting of the adrenergic response that was demonstrated to be sufficient to cause DCM under stress28. Whilst the contractile protein mutations in samples D1, D2 and D15 showed uncoupling, uncoupling was not a consistent feature of the TTNtv mutations suggesting that it is unlikely that uncoupling contributes to the DCM phenotype associated with titin truncating mutations.It is noteworthy that the DCM-associated mutations in contractile proteins are functionally distinct from DCM-related TTNtv mutant tissue. The faster relaxation rates of the contractile protein mutations D1, D2 and D15 suggest a reduced CaThese studies confirm the expectation that familial DCM caused by mutations in different genes can work through a number of different mechanisms that cause loss of function, in contrast to the apparent single mechanism for the gain of function in HCM.TTN isoform or altered titin phosphorylation levels20. In our fDCM and TTNtv samples we observed reduced passive stiffness with no difference in isoform expression  and no haploinsufficiency; nor was there incorporation of predicted truncated peptides into the myofibrils in common with other researchers19. There was no significant difference in overall titin phosphorylation level in the fDCM and TTNtv samples but we cannot exclude a change in the relative phosphorylation at sites in titin that enhance stiffness (PKC) and sites that reduce stiffness 29. At present we have no explanation for why the fDCM mutations cause reduced passive stiffness other than the possibility of reduced Ca2+-sensitivity may promote relaxation. For the TTNtv mutations it is possible that the mutations cause changes in exon usage that modulate elasticity as was observed with RBM20 ablation30 whilst causing no detectable change in molecular size. It is likely that the decreased passive stiffness would have an effect when the heart muscle is shortening under load that would not be apparent in the isometric contraction31. Such changes could be related to disruption of the dynamics of myosin head activation by stretch leading to reduced contractility32 or alteration of the “slow force response” by modulating of mechanosensitive signalling pathways that affect [Ca2+]i homeostasis and promote apoptosis and fibrosis as observed in our samples33. In concert such processes could impair contractility and potentially trigger a DCM phenotype.The most striking abnormality found in our samples was a 38% reduced passive stiffness for all the fDCM samples. In the case of the TTNtv mutations this was the only functional abnormality we detected and is thus likely to be indicative of the disease-causing abnormality. Our data showing a decrease in passive stiffness are in accord with other studies, notably of samples from idiopathic DCM hearts, and it has been proposed that this phenomenon could be explained by an increased expression of the N2BA 34. Twenty-eight patients with a diagnosis of familial or idiopathic DCM requiring a heart transplant at a young age were selected for sequencing and further studies of which 13 are listed in Supplementary Table 35. Patients were consented under ethical approvals obtained from the Australian Red Cross Blood Service (donor hearts) (HREC #2012/2814) and St Vincent’s Hospital, Darlinghurst, the University of Sydney , and the University of Kentucky, USA (08–03338-F2L). The collection and use of control samples used for immunofluorescence microscopy was approved by the ethics committee of the Medical Council of the State of Hessen. The investigations conform to the principles of the Declaration of Helsinki. The functional characteristics of some of the donor heart and myectomy samples have been previously reported36  and analyzed by pyrosequencing software.24. For the best resolution myofibril samples were dissolved in gel buffer without heating. High molecular weight proteins were separated on 4–15% SDS-PAGE gradient gels  with a running time of 2.5 hours at 100 volts or on 2% agarose/2% polyacrylamide “titin gels”.Whole tissue extracts were obtained for gel electrophoresis using T-Per Protein extraction reagent (Thermo Scientific 78510) including 1 µg/ml E-64, chymostatin and leupeptin protease inhibitors according to the manufacturer’s protocol. The myofibrillar fraction of heart muscle was prepared by our standard protocol38  and visualised with ECL plus .To assess protein phosphorylation 4–15% SDS-PAGE gradient gels were stained with Pro-Q Diamond phosphoprotein specific stain followed by SYPRO Ruby total protein stain. Western blots were made in a wet blotting apparatus for 5 hours at 250 milliamps at 4 °C. Blotted proteins were reversibly stained with MemCode Reversible stain kit (Thermo Scientific) and then probed with 9D10 monoclonal antibody to titin, specific to the I band region39. Agarose (0.3 g) was stirred in 20 mls of deionized water and dissolved by heating in a microwave for 1 min. The molten agarose was poured into a pre-warmed falcon tube (water bath set at 50 °C) and left to cool. The acrylamide solution was prepared as follows: 17.1 ml (deionized water), 3.6 ml 22.2% acrylamide , 4 ml gel buffer  and 15% ammonium persulphate, 200 µl (mixed in this order). The mixture was warmed in the water bath for 5 mins. 13.3 ml of the cooled agarose was added to the acrylamide solution and gently mixed, 30 µl of TEMED was added to the acrylamide/agarose mix and gently inverted. The gel solution was rapidly poured into a glass gel cassette (approx. 80 × 75 × 1.5 mm) and the combs were added instantly. The gels were then cooled at 4 °C for 7–10 min, then returned to room temperature for 2 hours. The samples were loaded and the gel was run at 4 °C at 7.5 mA for 30 mins, then raised to 15 mA .The method was slightly adapted from that of Tatsumi & Hattori40. TnI phosphorylation level in isolated troponin was measured by phosphate affinity SDS-PAGE as described by Messer et al.41.Troponin isolated from human heart samples was treated to change the level of phosphorylation. They were dephosphorylated by treatment with shrimp alkaline phosphatase  or phosphorylated by treatment with protein kinase A (PKA) catalytic subunit  as previously described2+-regulation of filament motility42. Thin filament movement over a bed of immobilised rabbit fast skeletal muscle heavy meromyosin (100 μg/ml) was compared in dual channel motility cells in which troponin varied by a single factor (mutation or phosphorylation state). The temperature was set to 29 °C. Filament movement was recorded and analysed as previously described43, yielding two parameters, the fraction of filaments moving and the speed of moving filaments. The fraction motile and the sliding speeds were measured over a range of Ca2+ concentrations to generate Ca2+-activation curves as shown previously44. The data were fitted to the 4-variable Hill equation to yield a value for EC50 and nH.Troponin isolated from human heart samples was treated to change the level of phosphorylation (see above). Thin filaments were reconstituted with 10 nM rabbit skeletal muscle α-actin , tropomyosin (40–60 nM) and troponin (60 nM) to study Ca45. Single myofibrils were isolated from samples of human left ventricular tissue45 and kept on ice for use in experiments for up to 3 days. Myofibrils were isolated from the human heart samples and suspended horizontally using specially prepared microneedles. Contraction and relaxation were initiated by a rapid Ca2+ concentration jump achieved by a fast-solution switch system implementing a double-barrelled micropipette moving across the mounted myofibril. Relaxing (0.01 µM Ca2+ or no added Ca2+) and activating (10 µM Ca2+) solutions contained (mM): MOPS 10, MgATP 5, free Mg2+ 1, DTT 5, phosphocreatine 10, creatine kinase (200 Units/mL), bacterial purine nucleoside phosphorylase (0.1 Units/mL), 7-methylguanosine 0.3, pH 7.0. The Ca-EGTA: EGTA ratio was set to obtain 10 mM total EGTA and the desired free [Ca2+]. Potassium propionate and sodium sulphate were added to adjust the ionic strength of the solution to 200 mM. The relaxing solution in the bath chamber was supplemented with (in μM): chymostatin 10, pepstatin 5, leupeptin 40, E-64 10, PMSF 200. The rate of force increase at 10 µM Ca2+, the maximum achieved isometric force, the rate of force regain after a quick release and re-stretch (kTR) and the slow and fast phase component rates of relaxation  at 0.01 µM Ca2+ were measured. Additionally, 30 mM of 2,3-butanedione monoxime (BDM) was added to the relaxing solution with no added Ca2+ during myofibril passive stiffness measurements. The experiments were performed at 17 °C.The apparatus and methodology for the measurement of force and passive stiffness in single myofibrils has been describedACT) and redevelopment (kTR) and for the fast phase of relaxation (kREL) were evaluated by curve-fitting using the equation LIN calculated from the slope value of the linear fit by normalizing it to the maximum tension , and the resected myocardial tissue did not present signs of ischemic damage or myocardial hypertrophy.in situ Cell Death Detection Kit  according to the manufacturer’s instructions. Tissue sections were examined by laser scanning confocal microscopy (Leica TCS SP2 and Leica TCS SP8). A series of confocal optical sections were taken using a Leica Plan Apo × 63/1.32 objective lens. After data acquisition, the images were transferred to a Windows 7 workstation for restoration and three-dimensional reconstruction using Imaris 7.7 multichannel image processing software  described below.5 μm cryosections were air dried and fixed with 4% paraformaldehyde. A rabbit polyclonal primary antibody against collagen I (Rockland) and a mouse monoclonal antibody against sarcomeric α-actinin  were used. Secondary detection system consisted of anti-rabbit or anti-mouse IgG and streptavidin linked Cy 3 (Amersham). TRITC Phalloidin was purchased from Sigma and used to stain F-actin; DAPI (nuclear staining) was purchased from Vector Laboratories. Apoptosis was analyzed using the 47. In brief, cryosections from at least two different levels in each tissue sample were used. All samples were immunolabeled simultaneously with identical conditions of fixation and dilutions of primary and secondary antibodies. Sections exposed to PBS instead of primary antibodies served as negative controls. For each heart at least 10 random fields of vision were analyzed using image analysis (Leica) and Image J software (NIH). The quantification process was performed blinded to the type of section, having on the screen only one channel showing F-actin labelling. For each quantification, a specific confocal setting was established and kept constant in all measurements. Quantification was performed by measurements of fluorescence intensity by using a range of 0 to 255 grey values. The area of collagen I was calculated as percent of positive labelling per tissue area. Quantification of apoptotic cells was performed as described48. Sarcomere length was measured by intensity profiles of α-actinin in XYZ axes using the three-dimensional extended “Section” mode of the Imaris 7.7 program.Quantification of collagen I was performed as described previouslySupplementary Information


Only limited information is available on the role of complement activation in malignant pleural mesothelioma (MPM). Thus, we investigated the circulating and tissue levels of the complement component 4d (C4d) in MPM. Plasma samples from 55 MPM patients, 21 healthy volunteers (HV) and 14 patients with non-malignant pleural diseases (NMPD) were measured by ELISA for C4d levels. Tissue specimens from 32 patients were analyzed by C4d immunohistochemistry. Tumor volumetry was measured in 20 patients. We found no C4d labeling on tumor cells, but on ectopic lymphoid structures within the tumor stroma. Plasma C4d levels did not significantly differ between MPM, HV or NMPD. Late-stage MPM patients had higher plasma C4d levels compared to early-stage (p = 0.079). High circulating C4d was associated with a higher tumor volume (p = 0.047). Plasma C4d levels following induction chemotherapy were significantly higher in patients with stable/progressive disease compared to those with partial/major response (p = 0.005). Strikingly, patients with low C4d levels at diagnosis had a significantly better overall survival, confirmed in a multivariate cox regression model . Our findings suggest that circulating plasma C4d is a promising new prognostic biomarker in patients with MPM and, moreover, helps to select patients for surgery following induction chemotherapy. With a median overall survival (OS) of 9 to 12 months following diagnosis, MPM remains associated with a fatal prognosis. Due to the rising incidence, accurate diagnosis and management of MPM are urgently needed5. Particularly, biomarkers that may facilitate the diagnostic process or indicate susceptibility to specific treatment are of utmost interest in order to improve survival.Malignant pleural mesothelioma (MPM) is a devastating malignancy with a strong link to prior asbestos exposure7. However, there is mounting evidence that the complement cascade is also involved in cancer development and progression11. One complement-related protein is the degradation protein C4d, a stable cleavage product of C4, accumulating following classical and lectin pathway activation14. C4d deposition in a tissue is regarded as an indirect proof of an activation of the complement cascade13. C4d is usually bound stably to the target structure but may eventually enter the circulation. Of note, C4d is routinely used as a tissue biomarker to investigate allograft tissue rejections in kidney transplants15. C4d has already been found to have a prognostic role in different types of cancer16. Ajona et al. found increased C4d levels both in plasma and bronchoalveolar lavage (BAL) samples from lung adenocarcinoma patients. Patients with elevated circulating as well as tumor tissue C4d levels had worse prognosis16. Furthermore, C4d expression has been shown to be associated with disease stage or poor prognosis in different other tumor types including astrocytoma and oropharyngeal squamous cell cancer17.The complement cascade is a major component of the innate immunity and it essentially contributes to immune surveillance, cell homeostasis and tissue regenerationet al. investigated a cohort of patients with or without pleural calcifications (PCs) due to prior asbestos exposure. C4 was only present in BAL samples of patients with PCs but not in those without PCs. Subjects with PCs were assumed to develop MPM less frequently18. Zerva et al. investigated several factors associated with humoral immunity in patients environmentally exposed to asbestos. Those patients had significantly increased serum C3 levels independently of the presence of PCs when compared to healthy controls. Although C3 is the target protein of activated C4, serum C4 levels did not differ between these groups19. Unfortunately the authors did not provide any data on the on the activation marker C4d. By analyzing mesothelioma pleural effusions, Nabil et al. demonstrated that complement factor H, a restrictive cofactor for the cleavage of C3b, potentially recruits monocytes and granulocytes to the malignant site via chemotactic function and thus supports malignant cell phagocytosis20.There is limited information about the role of the complement system in asbestos-exposed individuals and MPM development. In 2002, Galani Since the complement degradation product C4d has been shown to have a strong prognostic relevance particularly in lung adenocarcinoma, we investigated the tissue and circulating levels of C4d in MPM patients and compared these data with tumor load, chemotherapy response and clinicopathological parameters. Finally, we assessed the potential prognostic relevance of circulating C4d levels in patients with MPM.Clinical data and plasma samples of 55 consecutive, histologically verified MPM patients were collected at the time of diagnosis (n = 30) or before curative intent surgery after induction chemotherapy (n = 25) at the Medical University of Vienna between May 2011 and December 2014. In detail, all patient blood and tissue samples were retrieved before any diagnostic or curative intent surgical intervention or in case of post-chemotherapy samples before surgical resection. In consequence, acute clinical infections were ruled out at the time of sample collection. In 12 patients, plasma samples at the time of diagnosis as well as before surgery were available, representing pre- and post-chemotherapy samples. Of 32 of these 55 patients, formalin fixed and paraffin embedded (FFPE) tissue specimens were collected for immunohistochemistry analyses. Furthermore, plasma samples from an age-matched cohort of 21 healthy volunteers as well as from 14 patients diagnosed with non-malignant pleural diseases were included. Individual written informed consent was obtained from each individual and the study was performed after approval of the Ethics Committee at the Medical University of Vienna (#904/2009). Patients gave written informed consent to the inclusion of the material pertaining to them, that they acknowledge that they cannot be identified via the paper; and that they are fully anonymized. All methods included in this manuscript were performed in accordance with the relevant guidelines and regulations.All experiments that were reported in this manuscript were done complying with all mandatory laboratory health and safety procedures.Blood was collected via peripheral venous puncture from MPM patients and controls into 10 ml EDTA Vacutainers (BD Biosciences). Within 30 min after blood collection centrifugation was performed. Plasma supernatant was stored in aliquots at −80 °C until use.C4d and C3a fragments enzyme-linked immunosorbent assay (ELISA) kits were purchased from Quidel . For the analyses of C4d and C3a, samples were diluted 1:70 and 1:200, respectively, according to the manufacturers’ instructions. Standard curve generations and measurements in duplicates were performed according to the guidelines of the company.To obtain information about C4d presence within the tumor tissue and to compare these tissue levels with circulating C4d levels, 32 FFPE MPM tissue specimens were collected and analyzed by C4d immunohistochemical staining. Additionally, we performed immunohistochemical staining for C1q in 14 FFPE tissue specimens deriving from patients with high (n = 7) and low (n = 7) circulating C4d levels. After the sections were deparaffinized and rehydrated, endogenous peroxidase activity was blocked with 0.3% H2O2 in phosphate buffered saline. Heat-induced epitope retrieval was performed by using TRIS/EDTA buffer (pH = 9). Primary antibodies  were diluted 1:500 and 1:1000, respectively, and incubated for 60 min at RT. Antibody binding was detected with the UltraVision LP Detection System . Color development was achieved by DAB followed by hematoxylin counterstaining. Stainings were evaluated by a pathologist.21.In 20 of 55 patients, tumor volumetric measurement was available. Volumetric analyses were performed in patients with digitally available CT imaging data before and/or following chemotherapy. Chest CT images were analyzed by using dedicated software featuring semi-automatic segmentation with linear interpolation, allowing manual adjustments if necessary  - as previously describedIn summary, the segmentation and tumor volume quantification contained the following steps: the normal lung tissue, including bronchi and vessels, was marked semi-automatically by thresholding and region growing. Then pleural effusion and atelectatic lung areas were marked with a magnetic lasso function. After fixing normal lung tissue, pleural effusion, atelectatic lung and the outer part of the pleura was segmented semi-automatically. By using a lineal algorithm, interpolation between the marked slices was applied automatically. Finally, tumor volume was calculated by multiplying the sum of the voxels. Data was independently analyzed by two radiologists.12. Nevertheless, because only one patient had C4d plasma levels above 3 µg/mL at time of diagnosis, this cut-off value proved to be not suitable for MPM. However, when we analyzed circulating C4d values of patients with early- and late-stage disease, we found a distinct separation when using 1.5 µg/mL concentration as a potential cut-off. We also found that 1.5 µg/mL level was the best cut-off in our cohort by performing ROC curve analysis and Youden’s Index calculation for twelve months’ survival (data not shown). Hence, calculations described below were performed by using 1.5 µg/mL as cut-off.To investigate the role of circulating plasma levels of C4d, first we stratified our cohort by the cut-off of 3 µg/mL which was previously used in lung adenocarcinomaCategorical data was compared by performing Fishers’ exact or chi-square tests. Statistical differences between two groups were tested by Mann-Whitney U test. The significance of potential correlations of continuous parameters was investigated by using Pearson correlation. Overall survival (OS) was defined as time between initial MPM diagnosis and date of death or last follow-up. OS was estimated by the Kaplan-Meier method and a log rank test was used to calculate survival differences between two groups. A multivariate cox regression model was used to calculate hazard ratios and 95% confidence intervals for factors independently influencing OS. To investigate the accuracy of C4d as a predictor for survival and to distinguish between MPM and non-malignant pleural disease, Receiver Operating Characteristics (ROC) curve analysis was used. For each cut-off point, the Youden’s Index was calculated (sensitivity + specificity). All results were considered statistically significant when p < 0.05 two-sided. Analyses were performed using the SPSS Statistics 23.0 package  and GraphPad Prism 5.0.22.The clinicopathological parameters of the patient cohort are presented in Table In order to investigate C4d expression within MPM tumor tissue, we performed immunohistochemistry of 32 FFPE tissue specimens. Interestingly, we found no tumor cell specific C4d expression in these samples Fig. . TherefoIn order to investigate the relevance of circulating C4d as a potential diagnostic biomarker, we compared C4d plasma levels of MPM patients (n = 55), HV (n = 21) as well as of patients with NMPD (n = 14) Fig. . InteresWe also evaluated C4d values in terms of other main clinicopathological parameters including gender, age, smoking status and treatment regimen. Patients with epithelioid subtype, IMIG stage I/II, advanced age or those who underwent multimodality treatment were significantly more likely to have C4d levels below 1.5 µg/mL , high circulating C4d levels were significantly associated with a higher tumor load  after chemotherapy had significantly higher C4d levels compared to those with a partial or major response (PR/MR) .To investigate the prognostic relevance of circulating plasma C4d level, we dichotomized our cohort into patients with high C4d levels  and low C4d levels  at time of diagnosis. Patients with high plasma levels of C4d had significantly worse prognosis when compared to patients with low plasma levels  and low (n = 13) circulating C4d levels as well as in HVs (n = 12). In our cohort, plasma C3a levels were significantly higher in MPM patients compared to HVs  and high (n = 13) C3a categories by using the median plasma C3a level as cut-off (119 ng/mL). There was a tendency for decreased OS in patients with increased circulating C3a . The great majority of the resulting proteolytic cleavage product C4d remains at the side of formation and only a part is released as free C4d. As described above, C4d deposition was found in tumor samples not on cancer cells but on immune cells. To estimate whether the circulating C4d levels might be associated with the degree of tumor immune infiltration we selected 14 FFPE tissue specimens deriving from patients with high (n = 7) and low (n = 7) circulating C4d levels. Semiquantitative evaluation for tumor-associated inflammatory cells on hematoxylin-eosin stained sections revealed no correlation between circulating C4d levels and inflammatory cell infiltration (data not shown). C1q immunohistochemistry of the aforementioned 14 specimens revealed that tumors were negative for C1q in almost all samples  in comparison to normal mesothelial cell lines12, our study demonstrates that circulating C4d levels correlate with disease stage in MPM. Tumor tissue specific expression of C4d was shown to correlate with disease stage and tumor size in oral squamous cell carcinoma and astrocytoma17. However, no correlation analyses between local tumor load and circulating C4d levels were performed in any of these studies. Accordingly, our study is the first to show a correlation between circulating C4d levels and radiologically assessed primary tumor volume. Our results suggest that a more advanced disease as well as a higher local tumor load might lead to amplification in complement activation that might contribute to the opsonisation of malignant cells. We further hypothesize that not the MPM tumor cells themselves but - at least in part - the local immune infiltrative cells are responsible for triggering the classical complement activation pathway. Increased tumor load may lead to an increase in the absolute immune cell infiltration resulting ultimately in higher circulating C4d levels in advanced stages of MPM. However, additional factors might also contribute to the increased circulating C4d levels observed in advanced disease. Data deriving from lupus and atherosclerosis patients showed that complement activation can be triggered by circulating apoptotic and necrotic cells, recognized by complement initiators like C1q or CRP33. Therefore, increased numbers of circulating tumor cells or amount of tumor cell debris in advanced-stage patients possibly trigger the complement cascade. This assumption could explain the observed correlation between tumor burden and systemic levels of the complement-related degradation product C4d.As observed in lung adenocarcinomain vitro and in vivo experiments, are needed to identify the specific complement activation pathways that contribute to the progression of MPM.In order to study the specificity of circulating C4d as a marker for complement activation in MPM patients, we additionally measured plasma C3a levels. Similar to C4d, C3a is a complement-specific cleavage product that increases following complement activation. It can be induced by the classical, the lectin or the alternative complement activation pathway and is, accordingly, considered to be an unspecific surrogate parameter for complement activation. Plasma C3a levels were increased in MPM patients in comparison to HVs and there was a modest correlation between C3a and C4d levels. The prognostic power of C3a levels showed a similar tendency as C4d within the subcohort where both components could be measured. These findings further support the presence of complement activation in certain MPM patients and underline the clinical significance of complement activation in MPM. It is important to note that while the investigation of the additional markers C3a and C1q contributes to establishing the specificity of C4d as a biomarker, these correlative studies cannot dissect the upstream events and thus the specific mechanisms of complement activation in MPM. Further studies, including preclinical 36. To date, there exists no circulating biomarker in order to differentiate between histological MPM subtypes35. In our study, there was only a slight tendency for patients with non-epithelioid subtype to have higher C4d plasma values when compared to patients with epithelioid histology.Similar to C4d, other circulating biomarkers in MPM like mesothelin and activin A showed similar correlations with tumor load and stage of disease37. So far our study is the first showing circulating C4d levels to directly correlate with primary tumor load and chemotherapeutic response. Therefore, additionally to conventional response assessment - measuring C4d levels might facilitate the process of re-staging in order to estimate individual treatment efficacy more comprehensively.The majority of biomarker studies have focused on sample collections at the time of diagnosis and/or before surgery. In our study, we had the opportunity to analyse circulating C4d values in terms of individual chemotherapeutic response following induction chemotherapy. Remarkably, patients with progression after chemotherapy had significantly elevated circulating C4d levels when compared to those with a response following induction chemotherapy. Thus, measuring C4d levels may support the process of re-staging and re-evaluating of patients following induction chemotherapy. This is of importance, since MPM has a tendency to grow as a “rind” on the pleural surface and evaluating response and tumor expansion by RECIST criteria remains challenging


AbstractOver the last decade, inventorying and monitoring of marine biodiversity has significantly benefited from the active engagement of volunteers. Although several Citizen Science projects concern tropical reef ecosystems worldwide, none of the existing initiatives has yet specifically focused on their Mediterranean equivalents. Mediterranean coralline reefs, known as “coralligenous”, are bioherms primarily built by calcifying rhodophytes on hard substrates under dim-light conditions; they are considered hotspots of biodiversity and are extremely popular among divers due to their complex structure, conspicuous biological wealth and high aesthetic value. Nevertheless, data on their distribution, structure and conservation status is lacking for several Mediterranean areas while they are vulnerable to an increasing number of threats.In the framework of CIGESMED SeasEra (ERAnet) project a specialized Citizen Science project was launched, aiming to engage enthusiast divers in the study and monitoring of Mediterranean coralligenous assemblages through the gathering of basic information regarding their spatial occurrence, assemblage structure and associated pressures or threats. For its active implementation, a data collection protocol and a multilingual website were developed, comprising an educational module and a data submission platform. Georeferenced data reporting focuses on: (a) basic topographic and abiotic features for the preliminary description of each site, and the creation of data series for sites receiving multiple visits; (b) presence and relative abundance of typical conspicuous species, as well as (c) existence of pressures and imminent threats, for the characterization and assessment of coralligenous assemblages. A variety of tools is provided to facilitate end users, while divers have the choice to report additional information and are encouraged to upload their photographs. The long-term goal is the development of an active community of amateur observers providing widespread and ecologically significant data on coralligenous assemblages. Seawatchers). Although a plethora of CS projects deal with tropical coral reefs  of the Mediterranean Sea, through the integrated study of coralligenous assemblages. In the framework of CIGEDMED SeasEra project, a specialized CS initiative was launched aiming to engage enthusiast divers in the study of this Mediterranean habitat in order to obtain information regarding its spatial distribution, enable a primary characterization of the basic structure of its assemblages, and monitor potential pressures and threats to the habitat. The initial development of this pilot CS project was based on previous experience of the IMBBC  research group and CIGESMED partners on citizen science pilots design and implementation, as well as on the human, hardware and software resources of the LifeWatchGreece Research Infrastructure, a landmark in the European Strategy Forum on Research Infrastructures (ESFRI) roadmap. This paper aims to describe the background, basic principles and structure of the specialized CS initiative, along with the developed infrastructure, tools and guidelines that will support the ongoing collection of data on a Mediterranean scale.The Past experience from CS projects showed that it is relatively easy to design a CS approach and address volunteers but hard to ensure continuous production and flow of useful and reliable data . In retrA. Identification of new sites hosting coralligenous assemblagesThis is particularly important given the considerable paucity of spatial information regarding the presence of coralligenous bioherms in the eastern and southern Mediterranean regions, and the high cost of conducting extensive surveys to gather relevant data, since this habitat type usually develops in relatively deep waters (i.e. average of 20 m or even deeper in the eastern Mediterranean).B. Spatial recording of pressures and imminent threatsSuch baseline information is key in the design of effective management plans for the conservation of coralligenous assemblages in different parts of the Mediterranean.CIGESMED for divers CS initiative is to create a network of dedicated coralligenous observers spanning over the Mediterranean Sea. By creating a network of bilateral communication, scientists can address specific scientific / conservation issues that may arise in different areas  and gather valuable qualitative and semi-quantitative information with minimum cost. The majority of CS initiatives aim to maximize citizen engagement. However, 58% of the marine-related CS projects concentrate in easily accessible habitats such as beaches, estuaries, reefs and seagrass beds, which are found in waters shallower than 10 m , and are positively inclined towards using a platform that allows systematic sharing of their observations. Keeping these considerations aside, the designed CS module is openly addressing all amateur divers who are able and willing to contribute information on the occurrence of coralligenous bioherms, alongside specific information on conspicuous coralligenous species, existing pressures and threats, at any place they carry out their diving activities. In addition, the online platform could serve as an online repository for existing unpublished relevant data from various sources.Coralligenous bioherms usually develop in circalittoral waters, with their upper distribution limit ranging from less than 20 m depth in the western Mediterranean regions to more than 30 m in the eastern basin, although exceptions can be found in both areas depending on the prevailing environmental conditions . Given tFor the active implementation of the designed CS protocol, a portal supporting multilingual content was developed. It is currently available in 6 languages: English, French, Greek, Italian, Turkish, and Spanish. For the website development all available options were considered and Drupal® was selected as the platform that could optimally satisfy the demanding requirements of the designed website. Flexibility, stability and an API framework that can accommodate almost all conceivable functionality were the key characteristics for choosing the specific Framework - CMS (Content Management System). The website uses the Bootstrap 3.x, the most popular HTML, CSS and JS framework for development of complex dynamic websites and web applications, providing an easy way to adapt a responsive web design layout to the final template. All technologies involved in the development process are Open Source, based on the well-tested LAMP Platform  and most used in WWW. For map display functionality Leaflet library and the Openlayers 3.x were used combined with the Google Maps API v.3.divers, who can create, edit and delete content about observations, (b) country managers, being able to verify new user registrations, and (c) website administrators. Based on permissions set for those groups, each registered user has access to various features of the website. The demand of a complex observation registration form was covered by providing the user with the option to choose between different form displays when the user creates or edits an observation.An advanced user management system was developed, with three different groups of registered users: (a) Unity3D Platform and C# scripting language.A mobile application, that was originally developed in the framework of the LifeWatchGreece infrastructure, was modified and extended to support the present CS initiative. For this reason, a specialized "Citizen Science" sub-application was developed by utilizing Field trials for the implementation and optimization of the developed CS protocol took place in three countries: France (Marseille), Greece  and Turkey (Izmir). These included briefings to volunteer divers from local dive centres and associations, experimental dives for data collection followed by interviews, questionnaires and discussions, so as to come up with a more simplified list of requirements that clearly address the CIGESMED project objectives and make the data collection and reporting procedures as easy as possible for the participants. During these trials, the idea of developing an educational CS module to ensure a basic understanding of coralligenous bioherms and their associated communities also emerged, and was subsequently constructed.Preliminary data gathering was performed by the researchers and divers involved in CIGESMED project, thus allowing the assessment of coralligenous sites in four regions: Western Mediterranean, Ionian Sea, Aegean Sea and Levantine Sea.CIGESMED for divers website (http://cs.cigesmed.eu/) comprise:The developed CS methodological protocol and in situ data collection in five languages; English  and information on their diving profile (i.e. diving experience and certification level), dive computer brand and type (for standardizing temperature data records), past experience from any other CS projects, area of taxonomic interest in cases of professional scientists and enthusiast naturalists, and dive centre Fig. . Users aThe main data submission infrastructure (dive slate and web version) provides fields for reporting information relevant to the characterization of the site and the assessment of its coralligenous assemblages, organized in two sections: (a) basic data and parameters, and (b) ecological observations Fig. . In the This section integrates typical geographic and topographic data along with several easily assessed abiotic environmental parameters which can allow a preliminary description of the site, as well as the development of data series for sites receiving multiple visits Table . The locin situ by amateurs were omitted in favour of more conspicuous ones. On the other hand, the inclusion of a free-text field for reporting organisms not included in the basic list, allows more advanced users to upload additional records according to their best knowledge level. Information on the appropriate underwater recording of species, as well as their importance for the marine ecosystem are presented in the multilingual guidelines in a comprehensive manner.This section of the CS platform focuses on: (a) the presence and relative abundance of typical conspicuous species of coralligenous assemblages and (b) the recording of pressures and imminent threats. A total of 23 taxa belonging to 7 taxonomic groups were included in the CS protocol . As the number of observations is directly linked to diver’s experience and knowledge of the marine realm, 5 levels were assigned to male  and female (Oceanids and Nereides) ancient Greek sea deities . All participants and contributors, including dive and nature associations and clubs are accredited and, in certain cases, could be considered as co-authors in relevant scientific and mass media publications (see acknowledgements and author contributions sections).CIGESMED for divers website in real time. The application showcases a simplified version of the observations submitted to the "CIGESMED for divers" website on a map. Users are given the option to select and view details for each observation, such as the list of reported species  through images, videos and 3D models that can be virtually manipulated, dissected and rotated using online tools  and receives data in JSON format from ies Fig. . By seleies Fig. . FurtherThe engagement of citizens in marine research is particularly important, since professional scientific activities are often restricted by available financial and human resources . MoreoveThe objectives of this CS initiative extend beyond single-point data collection by providing a way to monitor coralligenous assemblages and assess potential degradation through the creation of long-term data series in sites receiving multiple visits (e.g. Marine Protected Areas). The inclusion in the protocol of several rare, protected, and commercial species , could further assist in their monitoring and conservation. This is particularly important in countries where research funds dedicated to environmental assessment and monitoring of the marine environment are low or non-existent; under such circumstances the contribution of skilled citizen scientists could critically enable the persistent collection of qualitative or semi-quantitative data .ELNAIS).The increasingly important role that active volunteers can play in reporting of the presence and spread of marine allochthonous species has been recently pointed out , along wCIGESMED for divers CS project for the coralligenous habitats in the Mediterranean. It also provides the electronic infrastructure needed for: (a) the data management in order to be harvested by the international aggregators, such as OBIS and GBIF; (b) the appropriate electronic infrastructure in order to analyze the data and interpret the results, in the form of the virtual laboratory RvLab . The provision of detailed guidelines, easy-to-follow directions, high quality photos and simplified dive-slate versions of the CS protocol offer essential tools for data standardization and minimization of observer bias. Additionally, the online data submission platform encourages divers to upload their photos for each species record, thus facilitating quality control. Each observation is associated to the user’s name, giving the opportunity to the CS research team to access the profile features of the diver  or even to contact him/her directly in exceptional cases . For this reason, we have established groups of "country managers" among the involved researchers, acting as focal points responsible for providing support to users, managing and evaluating observations from different geographic regions, performing quality control and contacting divers in their language when needed.Sharing data is one of the main objectives of CIGESMED. However, data assembled by non-experts should go through quality control before being used in scientific analyses or reposited to global biodiversity databases . The lisCIGESMED for divers project will be further used to: (a) calculate the values of indices for the assessment of the "good environmental status" (GES) of the habitat, as required by the Marine Strategy Framework Directive (MSFD). For this purpose, only indices requiring a minimum of information will be used, such as those proposed under the Descriptor 6 of the MSFD (sea-floor integrity)  ; (b) thetegrity)  that canDuring the field trials and discussions with participants, their associations and diving clubs, as well as national MPA authorities, it became evident that a considerable proportion of divers, especially in the eastern Mediterranean, were not aware of the existence and importance of coralligenous assemblages despite the fact that these formed their preferred diving spots. The educational module of the project (i.e. guidelines and website) combined with the experience gained from the participation of divers directly raises public awareness. Experience from the field trials and past CS projects showed that knowledge exchange between citizen scientists and researchers promotes trust and facilitates application of marine conservation schemes . ParticiCharismatic, flagship species  that thrive in coralligenous bioherms can raise the interest of recreational divers and greatly increase their awareness for the habitat as a whole, or their willingness to invest time for data recording. For example, in a recent citizen science project regarding the presence of red coral along the coasts of Italy, A recent review study by CIGESMED for divers CS protocol, these activities, are expected to enhance the creation of long-term data series, increase public awareness and establish communication and knowledge exchange on a more trust-worth and permanent basis.A series of joint activities organized by the CIGESMED CS researchers, with selected motivated diving associations and MPA management authorities have been scheduled in Greece , France , and Turkey . Those include photo-sampling of permanent quadrats, recording of environmental data (e.g. through the deployment of loggers) and recording of threats and impacts on a scheduled and systematic basis. Joint missions will further include training of the involved divers and demonstrating the proposed activities using the developed CS protocol and e-infrastructure. Furthermore, online photo-contests are to be periodically scheduled and high-quality photographs uploaded by users will be screened on the homepage of the website or could be used in future scientific and educational publications, always acknowledging the photographer. Combined with the Supplementary material 1CIGESMED for divers – Citizen Science for CIGESMEDData type: GuidelinesBrief description: Guidelines for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82405.pdfGatti G., Thierry de Ville d’Avray L., David R., Dimitriadis C., Gerovasileiou V., Dailianis T., Sini M., Salomidi M., Dogan A., Issaris Y., Cinar M.E., Koutsoubas D., Arvanitidis C., Feral J-P.Supplementary material 2CIGESMED pour les plongeurs – Les sciences participatives pour CIGESMEDData type: GuidelinesBrief description: Guidelines for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82406.pdfGatti G., Thierry de Ville d’Avray L., David R., Dimitriadis C., Gerovasileiou V., Dailianis T., Sini M., Salomidi M., Dogan A., Issaris Y., Cinar M.E., Koutsoubas D., Arvanitidis C., Feral J-P.Supplementary material 3CIGESMED για δύτες, πολίτες-επιστήμονες για το πρόγραμμα παρακολούθησης των κοραλλιγενών οικοτόπωνData type: GuidelinesBrief description: Guidelines for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82408.pdfGatti G., Thierry de Ville d’Avray L., David R., Dimitriadis C., Gerovasileiou V., Dailianis T., Sini M., Salomidi M., Dogan A., Issaris Y., Cinar M.E., Koutsoubas D., Arvanitidis C., Feral J-P.Supplementary material 4CIGESMED per i subacquei - Citizen science per CIGESMEDData type: GuidelinesBrief description: Guidelines for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82410.pdfGatti G., Thierry de Ville d’Avray L., David R., Dimitriadis C., Gerovasileiou V., Dailianis T., Sini M., Salomidi M., Dogan A., Issaris Y., Cinar M.E., Koutsoubas D., Arvanitidis C., Feral J-P.Supplementary material 5Dalgıçlar için CIGESMED – CIGESMED için Vatandaş BilimiData type: GuidelinesBrief description: Guidelines for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82411.pdfGatti G., Thierry de Ville d’Avray L., David R., Dimitriadis C., Gerovasileiou V., Dailianis T., Sini M., Salomidi M., Dogan A., Issaris Y., Cinar M.E., Koutsoubas D., Arvanitidis C., Feral J-P.Supplementary material 6CIGESMED for divers – Citizen Science for CIGESMEDData type: SlatesBrief description: Printed fill-in forms for underwater observation for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82412.pdfGatti G., David R., Dimitriadis C., Doğan A.Supplementary material 7CIGESMED pour les plongeurs – Les sciences participatives pour CIGESMEDData type: SlatesBrief description: Printed fill-in forms for underwater observation for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82413.pdfGatti G., David R., Dimitriadis C., Doğan A.Supplementary material 8CIGESMED για δύτες, πολίτες-επιστήμονες για το πρόγραμμα παρακολούθησης των κοραλλιγενών οικοτόπωνData type: SlatesBrief description: Printed fill-in forms for underwater observation for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82419.pdfGatti G., David R., Dimitriadis C., Doğan A.Supplementary material 9CIGESMED per i subacquei - Citizen science per CIGESMEDData type: SlatesBrief description: Printed fill-in forms for underwater observation for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82420.pdfGatti G., David R., Dimitriadis C., Doğan A.Supplementary material 10Dalgıçlar için CIGESMED – CIGESMED için Vatandaş BilimiData type: SlatesBrief description: Printed fill-in forms for underwater observation for the implementation of CIGESMED Citizen Science protocol for the study and monitoring of Mediterranean coralligenous assemblages.File: oo_82417.pdfGatti G., David R., Dimitriadis C., Doğan A.


Over the past decade, it has been convincingly shown that regularly performing repeated brief supramaximal cycle sprints  is associated with aerobic adaptations and health benefits similar to or greater than with moderate-intensity continuous training (MICT). SIT is often promoted as a time-efficient exercise strategy, but the most commonly studied SIT protocol  takes up to approximately 30 min per session. Combined with high associated perceived exertion, this makes classic SIT unsuitable as an alternative/adjunct to current exercise recommendations involving MICT. However, there are no indications that the design of the classic SIT protocol has been based on considerations regarding the lowest number or shortest duration of sprints to optimise time efficiency while retaining the associated health benefits. In recent years, studies have shown that novel SIT protocols with both fewer and shorter sprints are efficacious at improving important risk factors of noncommunicable diseases in sedentary individuals, and provide health benefits that are no worse than those associated with classic SIT. These shorter/easier protocols have the potential to remove many of the common barriers to exercise in the general population. Thus, based on the evidence summarised in this current opinion paper, we propose that there is a need for a fundamental change in focus in SIT research in order to move away from further characterising the classic SIT protocol and towards establishing acceptable and effective protocols that involve minimal sprint durations and repetitions. Addressing the negative consequences associated with the high prevalence of physical inactivity in the general population  is one o2max), this specific protocol was used by more than half of all included studies  and [AMP], ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leaking [2max is generally considered to be limited by central factors [2max due to increased mitochondrial density [It is generally accepted that greater volumes/higher intensities of MICT will lead to more pronounced cardiometabolic adaptations –52, ther leaking , etc.).  factors , recent  factors , and sev density , 57, 58. density , but cou density .2max [2max.Potential mechanisms have been investigated in HIIT studies , 61, 62,}V˙O2max , 76 doesTo date, the limited amount of available data neither supports nor refutes that the classic 4–6 × 30-s SIT protocol will lead to more pronounced activation of potential signalling pathways involved in any of the cardiometabolic adaptations to SIT compared with shorter/easier protocols. It is entirely plausible that the severe disruption of homeostasis associated with supramaximal exercise rapidly ‘saturates’ the signalling response and that regularly performing just a few brief supramaximal sprints is sufficient to gain the desired health benefits.2max with the 10-s protocol. This finding was supported a few years later by Zelt et al. [2max response to the classic SIT protocol with 30-s sprints (4%) and a modified protocol with 15-s sprints (8%). These findings are important as the duration of supramaximal sprints has a substantial impact on perceived exertion; the strong contribution of phosphocreatine hydrolysis to energy demands during the first approximately 10 s of a 30-s Wingate sprint means that fatigue during this phase is relatively low, whereas the gradual switch to glycolysis as the predominant energy source [A growing body of evidence supports that performing fewer and/or shorter sprints does not impair the cardiometabolic adaptations associated with SIT Table . Hazell t et al. , who repy source  is assoc2max following a protocol involving three repeated 30-s Wingate sprints; however, this protocol involved 20 min of passive recovery following each sprint, negating its potential time efficiency. More recently, Ijichi et al. [2max (+14% following 4 weeks of training).Similar to reducing sprint duration, reducing the number of sprint repetitions will also decrease the perceived exertion associated with SIT. As mentioned in Sect. i et al.  also use2max by 10–13% [2max (+12%) and reported a 7% decrease in mean arterial pressure [2max does not plateau after 6 weeks (+12% after 6 weeks and +19% after 12 weeks), and that the increase in insulin sensitivity in response to REHIT (+53%) is not significantly different compared with the increase with MICT (34%) [In our own laboratory, we have demonstrated that 6 weeks of 3-weekly 10-min SIT sessions involving just two 20-s Wingate sprints   .2max [2max. Although the magnitude-based inference of this effect was ‘possibly small’, and therefore further research is required to provide a definitive answer to the question as to whether performing more sprints is worse, this question is irrelevant for the practical implications of our finding; performing more sprints was clearly not better for improving 2max. The logical question then is whether regularly performing just a single supramaximal sprint will be sufficient to improve 2max; however, in two recent studies we have provided initial data to suggest that this is not the case [The strongest evidence in favour of the efficacy of shorter/easier SIT protocols comes from our recent meta-analysis in which we examined the modifying effect of the number of sprint repetitions in an SIT session on the increase in }V˙O2max . A surprthe case , 76. It In recent years, the focus of research investigating time-efficient alternatives to current exercise recommendations has shifted away from the classic SIT protocol towards HIIT protocols. Gibala’s HIIT protocol involving 10 repeated 1-min sprints at approximately 90% of maximal heart rate interspersed with 1 min recovery , 79, 80,In a recently published debate on the pros and cons of HIIT as a public health strategy , the pro