Staphylococcus aureus was conducted to validate the antibiofilm capacity of the peptide-modified stainless steel samples. XPS analysis confirmed that the optimal dopamine concentration was 40 µg ml−1 in the coupling reaction. Element analysis showed that dopamine and the peptides had bound to the steel surfaces. The robustness assay of the modified surface demonstrated that most peptide molecules had bound on the surface of the stainless steel firmly. The contact angle of the modified surfaces was significantly changed. Modified steel samples exhibited improved antibiofilm properties in comparison to untreated and dopamine-only counterpart, with the peptide 1 modification displaying the best antibiofilm effect. The modified surfaces showed antibacterial capacity. The antibiofilm capacity of the modified surfaces was also surface topography sensitive. The steel sample surfaces polished with 600# sandpaper exhibited stronger antibiofilm capacity than those polished with other types of sandpapers after peptide modification. These findings present valuable information for future antifouling material research.Biofilm formation on surfaces is an important research topic in ship tribology and medical implants. In this study, dopamine and two types of synthetic peptides were designed and attached to 304 stainless steel surfaces, aiming to inhibit the formation of biofilms. A combinatory surface modification procedure was applied in which dopamine was used as a coupling agent, allowing a strong binding ability with the two peptides. X-ray photoelectron spectroscopy (XPS), elemental analysis, contact angle measurement and surface roughness test were used to evaluate the efficiency of the peptide modification. An antibiofilm assay against  In many cases, this colonization leads to the subsequent formation of biofilms. Biofilms result from the accumulation of organic molecules, microorganisms and metabolites and are ubiquitous on surfaces submerged in aqueous environments . The ratet al. obtained a material with low surface energy by reacting a polypeptide with stainless steel [et al. proved that the peptide–steel reaction led to a formal or semi-formal organic–metal covalent bond formation, because stainless steel shared electrons with the disulfide bond of the peptide. The antibiofilm ability of the peptide-treated steel was also improved due to surface physiochemical properties of the material [et al. expressed the hydrophobic membrane proteins of Bacillus subtilis in Escherichia coli using recombinant genetic engineering techniques, which can be used to produce peptides for modifying surfaces [et al. [Stainless steel is widely used in many fields, including medical device, food processing and ship manufacture industries. Bacteria and organic residues can easily adsorb onto stainless steel surfaces leading to biofouling . Surfacess steel . Anotherss steel . Davis ematerial . Yang etsurfaces . Ren et  [et al.  reacted Dopamine coating has recently emerged as an extremely attractive approach for single or two-step surface modification of almost all kinds of materials . DopaminStaphylococcus aureus (S. aureus) were evaluated. The relationship between the surface topography and the antibacterial/antibiofilm properties was correlated through multiple surface characterization assays.In this study, a combinatory surface modification procedure (two-step reaction) was investigated in which dopamine was used as the coupling agent. Two types of synthetic peptides were sequentially bound to dopamine-treated 304 stainless steel surfaces to investigate the benefits of dopamine on the peptides' binding. The impacts of the two peptide-modified metal surfaces on the adhesion of 2.2.1.AISI 304 grade annealed stainless steel sheets with a thickness of 250 µm were purchased from Wuhan steel factory, which was studied for marine facilities and medical implants –20. Stai2.2.–1. The polished and cleaned stainless steel discs (SS) were immersed in dopamine solutions. The dopamine treatment was performed at room temperature for 24 h with rotary shaking at a rate of 60 r.p.m. At the end of the reaction, dopamine-treated discs, denoted as SS-D, were removed from the reaction mixture and ultrasonically washed for 20 min in deionized water, followed by rinsing with deionized water to remove free polymeric dopamine and air-dried. The SS-D was stored at room temperature for characterization and further reaction with peptides.Different concentrations of dopamine solutions were prepared by dissolving dopamine in Tris–HCl buffer  at room temperature into final concentrations of 5, 10, 20, 40, 80 and 160 µg ml2.3.−1 in Tris–HCl buffer, pH = 8.5) was added into 24-well plates for binding peptides. The reaction was allowed to proceed at room temperature with continuous stirring (60 r.p.m.) for 24 h. The reacted discs (SS-P) were removed from the reaction mixture and rinsed with a large amount of deionized water. The surfaces were stored in a dry and dark place before subjecting to property characterization and antibiofilm tests.The SS-D discs were placed into 24-well plates (one sample per well) and 1 ml peptide solution . The contact angle was measured within 15 s for a water drop (1 µl) on the surfaces and the mean of five measurements was reported. The elemental information of the sample surfaces was further analysed by X-ray photoelectron spectroscopy . The resolution of the instrument was 0.45 eV for all elements except for He, and the scan step was 0.12 eV. Each sample was scanned 12 times, and the final XPS spectra were generated by the software within the instrument.Sa) and root mean square height (Sq) were measured and calculated to describe surface microstructures of samples and the amplitude/height distribution of the surface. Sa gives a good general description of height variations; Sq represents the standard deviation of the distribution of surface heights. The lateral and vertical resolutions for the surface profile measurement were 0.1 and 0.2 µm, respectively. The sampling area was selected as 0.8 × 0.8 mm and each step of the scan was set to 0.02 µm. The measurements were collected in three different locations for each sample. The surface parameters were derived from the collected data by the calculation programme of SPI. Ultra-deep three-dimensional micro-system (VHX-2000), which has a large depth of field, was used to record the topography of the polished discs.The surface textures were analysed by the LI-3 surface profile measuring instrument . Two key surface roughness parameters, roughness average (2.5.S. aureus (ATCC 12600) and E. coli (ATCC PTA-10989), the most common biofilm-producing Gram-positive and Gram-negative organisms and being extensively studied for marine environment and biomedical implants previously [S. aureus and E. coli strains were cultivated in TSB medium  and LB broth , respectively. Bacterial strains (1 × 106 CFU ml−1) were cultivated with three consecutive batch cultures at 37°C with shaking (180 r.p.m.). Experiments were conducted using cultures at stationary phase.The two bacterial strains, eviously –24, were8 CFU ml−1 and 1 ml well−1) were added to the wells. Plates were covered and sealed with Parafilm®. Samples were incubated at 37°C with continuous shaking (60 r.p.m.) for 16 h. Medium and planktonic/non-adhered bacteria were removed and plates were rinsed at room temperature three times with deionized water. In total, 300 µl of 0.1% crystal violet solution was added to each well. Plates were stood for 10 min for staining. Crystal violet solutions were discarded after staining and the discs were washed six times using sterilized water. Discs were air-dried for 1 h in a laminar flow hood. The total stain was quantified by the extraction of crystal violet by addition of 1 ml 30% acetic acid per well. Absorbance values of sample elutions were recorded at 590 nm by a plate reader . For testing the durability and robustness of the peptide-modified surfaces, SS-P1 and SS-P2 were immersed in distilled water for 2, 12, 24, 48 and 96 h with a gentle shaking (20 r.p.m.), respectively. The samples were air-dried and then were used to conduct the antibiofilm assay.To conduct bacterial adhesion tests, samples  were put into 24-well suspension culture plates (one sample per well). Bacterial solutions (1 × 105 CFU ml−1 in PBS and TSB). After incubation at 37°C for 3 h, samples were washed three times with autoclaved water. Samples were then immersed in 2 ml sterilized PBS solutions and sonicated for 5 min. Decimal dilutions (10−1 and 10−2) of the resulting bacterial suspension were each spread onto TSA  plates. After incubation for 24 h at 37°C, the number of colonies was counted. Untreated sample was used as a control. The antibacterial property was evaluated according to the following equation:E is the antibacterial efficiency; N1 is the number of bacteria on untreated samples; N2 is the number of bacteria on peptide-modified surfaces.To conduct the bactericidal test, bacterial solutions were centrifuged (5000 r.p.m. for 20 min) and re-suspended in PBS solution (pH 7.4) and TSB medium, respectively. Sample surfaces were covered by 100 µl bacterial solutions . 3.3.1.Pseudomonas aeruginosa (Pa), can react with metals and shows antimicrobial properties [The 16-mer peptide 1 (P1)  with an operties . P1 was 3.2.Dopamine was used as a coupling agent that was bound to the metal disc first, and then tethered to antibacterial peptides. Dopamine concentration affected the degree of reaction and resulted in different surface modifications. 304 stainless steel reacting with dopamine under different concentrations was studied first. It was found that, after reaction with dopamine, all treated surfaces of the discs were covered by dark or brown-coloured layers. The darkness increased considerably at higher concentration of dopamine and most thick and dark layers were easily removed during the rinsing step.a show that there were no Fe2p peaks (around 711 eV and 724 eV) in the samples when used dopamine concentrations were larger than 10 µg ml−1, while a small peak at around 711 eV appeared in the sample reacting with 5 µg ml−1 dopamine. This suggested that the dopamine-grafted layer was beyond 10 nm thickness and no iron signal underneath was detected for the samples reacting with dopamine solutions in concentrations larger than 10 µg ml−1. Figure 1b shows the characteristic peaks of N1s, and it can be seen that the peak intensities in the samples were dopamine concentration-dependent. The N1s peaks of SS-D XPS spectra shifted when compared with untreated samples. The N1s peaks at 399.8–400.2 eV were due to the presence of amine groups and the intensities of the peak increased with the increase of the used dopamine concentration then reduced when the dopamine concentration increased further. Differences in binding energies were ascribed to differences in dopamine oxidation intermediates.To confirm the presence of dopamine polymerization products on the treated discs, XPS spectra of sample surfaces were collected to quantify elemental nitrogen. The XPS survey spectra in figure 1−1. XPS examination of the content elemental nitrogen on the samples confirmed this observation (as shown in figure 2a). Nitrogen percentage in dopamine and dopamine oxidation intermediates was around 9.1–10%, which was consistent with the results generated by XPS analysis. The highest value of nitrogen content was detected in the samples reacting with dopamine solution at a concentration of 40 µg ml−1.It was apparent that the amount of dopamine bound to substrate materials depended on dopamine concentration. The bound dopamine reached maximum when its reaction concentration reached 40 µg mlb). As dopamine is a hydrophilic molecule, the contact angle of surfaces would decrease if the surface was bound by dopamine. The contact angle of untreated sample was 65.7°, while contact angle of SS-D was about 40° when using dopamine solutions with concentrations larger than 10 µg ml−1 (b). It was confirmed that the optimal reaction concentration was about 40 µg ml−1. Consequently, the following experiments used dopamine solutions at a concentration of 40 µg ml−1.Bound dopamine was detected indirectly by the contact angle measurement. After reaction with dopamine, a decrease of water contact angles of the stainless steel surfaces was observed b. As dop µg ml−1 b. It was3.3.Contact angles of untreated SS-D and SS-P samples are presented in 3.4.3.4.1.Staphylococcus aureus and E. coli are common biofilm-producing Gram-positive and Gram-negative bacteria, respectively, and are ubiquitous in nature. To assess the ability of these species to undergo bacterial adhesion and biofilm formation, untreated discs were immersed into S. aureus and E. coli solution at 37°C with gentle shaking (60 r.p.m.) for 16 h. After the incubation, the samples were stained using crystal violet. Optical images were obtained and shown in E. coli (b) and S. aureus (c). As crystal violet intensity is proportional to the quantity of biofilm, the images revealed that the S. aureus strain had greater biofilm forming ability on the surface of stainless steel than the strain of E. coli. As S. aureus had a better capacity for the formation of biofilm in the study, it was used in the following bacterial adhesion assays.A layer of pure biofilm formed on the stainless steel sample surface incubation with E. coli b and S. . aureus c. As cry3.4.2.S. aureus, the transparency of remaining bacterial solution within plates changed from clear to turbid in different groups as shown in After incubation with In this study, the robustness assay of the modified surface has been taken to test the stability of the resultant modified surfaces and associated antibiofilm capacity . The OD 3.4.3.S. aureus was assessed by counting bacteria colony  showed that the sample texture following polishing with fine sandpaper was significantly smoother than that by coarse sandpaper. The two surface profile parameters, Sa and Sq, calculated by SPI (c) further confirmed that discs polished with 2000# sandpaper were smoother than those polished with smaller size papers. Three-dimensional morphology models were also obtained by simulation software of SPI (b). The 600# sandpaper-polished surface seems to possess the proper pattern to bind more peptides and consequently reduce bacterial attachment more efficiently. The size of each individual S. aureus is approximate 0.8 µm, which is slightly larger than the value of Sa of 600# sandpaper-polished surface. This result was consistent with attachment point theory [The ultra-deep three-dimensional micro-system images and SPI-simulated image models a,b showed by SPI c furthere of SPI b. The 60t theory ,27.Figu4.Although biomaterials yielded by reaction of peptides with metals have been reported previously ,29, low −1. This was because, at higher concentration, the self-polymerization reaction of dopamine would be dominated, which prevented the formation of stable binding of dopamine to metal surfaces. Both dopamine and PDA are hydrophilic molecules, and dopamine-treated surfaces became hydrophilic even if there was only PDA on the surface. That may explain the similar and low contact angle values for all dopamine-treated discs when dopamine concentrations were greater than 10 µg ml−1 (b).During the coupling reaction with dopamine, poly-dopamine (PDA) was generated by the oxidation and self-polymerization reaction of dopamine in weak alkaline solution under an oxygen-enriched environment . The acc µg ml−1 b.XPS analysis was confirmed as a sensitive tool to study surface modification and distinguish the different surface treatments by analysis of S2p orbitals of sample surfaces. There were no classical sulfur electron orbitals in stainless steel and dopamine. The strong peaks that appeared at 160 eV region of the SS-P1 and SS-P2 spectra suggested that peptides were bound on the surface .The contact angle, which evaluates the wettability of the sample surfaces, can be used to assess the success of surface modification. After dopamine treatment, the contact angle of stainless steel was decreased because dopamine possesses good hydrophilicity. The contact angle increased after modification by two peptides, which indirectly indicated that peptides have bound to the metal surfaces because the peptides have lower hydrophilicity . P2-treaS. aureus used in this study showed a stronger biofilm formation ability on stainless steel surface than the strain of E. coli , serine (S) and threonine (T) residues could bind into the PDA layer via covalent bonds –40. PDA/ium spp.  should b5.via dopamine coupling. Dopamine formed a stable binding on the metal surface, and acted as a coupling agent to improve binding efficiency of the peptides on the surface of the stainless steel. When using the optimal concentration of dopamine solution (40 µg ml−1) for peptide modification of the metal surface, the samples treated with dopamine and peptides possessed antimicrobial capacity. SS-P1 was the best, followed by SS-P2, and SS-D was the last. Surface topography of the metal sample affected the adhesion capacity of bacteria on the surface of the material. The antibiofilm property of the sample surfaces polished by 600# sandpaper was best in S. aureus solution. Hence, the combination of peptide surface modification and topographic alteration on stainless steel materials can be a promising technique to introduce an antibiofilm feature to metal surfaces.In summary, stainless steel surface was successfully modified by two types of designed peptides


Dissolved organic matter (DOM) strongly affects water quality within boreal forest ecosystems. However, how the quality of DOM itself changes spatially is not well understood. In this study, to examine how the diversity of DOM molecules varies in water moving through a boreal forest, the number of DOM molecules in different water samples, i.e., rainwater, throughfall, soil water, groundwater, and stream water was determined using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in Norway spruce and Scots pine stands in eastern Finland during May and June 2010. The number of molecular compounds identified by FT-ICR MS (molecular diversity) ranged from 865 to 2,194, revealing large DOM molecular diversity in the water samples. Additionally, some of the molecular compounds were shared between different water samples. The DOM molecular diversity linearly correlated with the number of low-biodegradable molecules, such as, lignin-like molecules (lignins), but not with dissolved organic carbon concentration. The number of lignins shared between different sampling locations was larger than that of any other biomolecular class. Our results suggest that low-biodegradable molecules, especially lignins, regulate spatial variations in DOM molecular diversity in boreal forests. Dissolved organic matter (DOM) plays a central role in changes in water chemistry within boreal forest ecosystems. It affects the acidity and color of the water that passes through the tree canopy or forest floor1234678911Boreal forests contain more than one third of the carbon retained in forest vegetation and soils globally15et al.Recent progress in the chemical characterization of DOM has enabled us to obtain an overall picture of molecular alterations/transitions of DOM in water collected from forests. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is one of the most innovative methods and allows us to detect different types of molecules in DOM based on the cyclotron frequency of charged ions in a magnetic field. This method has provided reproducible and reliable data concerning the mass spectrum of DOM in river and marine water samples, which consist of more than a hundred different types of DOM molecules22232427The objectives of this study were (1) to evaluate variations in molecular diversity and the molecular compounds divided into specific biomolecular classes in rainwater passing through a boreal forest using FT-ICR MS and van Krevelen diagrams, and (2) to examine how sampling location affects DOM molecular compounds in a boreal forest catchment in eastern Finland.The number of molecular compounds detectable by FT-ICR MS  and subsequently the number of each biomolecular class vary with water movement .The molecular compounds identified as the same DOM compound exist at different sampling locations.DOM molecular diversity increases with increasing DOC concentration.Our hypotheses were as follows:p > 0.05).The number of DOM molecular compounds detected as m/z peaks (molecular diversity) was 1,293 ± 307 SD on average across all of the water samples and ranged from 865 to 2,194 . The coeEach DOM molecular compound was assigned to one of seven biomolecular classes , unsaturated hydrocarbons (UH), lignin-like molecules (lignins), tannin-like molecules (tannins), or condensed aromatic structures (CAS)), or it was noted that it could not be assigned to any class. Our results revealed that the number of lignins was larger than that of any other class and only a few molecules of As/Ch were found in every sample except the bulk deposition sample obtained in May . The numr = 0.83, p < 0.001 in May, r = 0.77, p < 0.001 in June; 3b: r = 0.86, p < 0.001 in May, r = 0.77, p < 0.001 in June; 3c: r = 0.77, p < 0.01 in May, r = 0.76, p < 0.001 in June). The proportion of the sum of lignins, tannins, and CAS in the molecular diversity in soil water was generally high (28–56%) compared with those in bulk deposition and throughfall (20–31%), although not in comparison to the throughfall in June (56%).DOM molecular diversity increased significantly with increasing numbers of lignins, tannins, and CAS in both May and June . There was no correlation between DOC concentration and DOM molecular diversity .The DOC concentration across all of the water samples was 11.2 ± 10.4 mgC l mgC l–1 . The CV The number of molecular compounds detected as the same m/z peak between different water samples ranged from 153 to 1010. The proportion of those compounds in the molecular diversity ranged from 10% to 60% depending on sampling location. Additionally, the number of lignins shared between different water samples was larger than that of any other biomolecular class and ranged from 52 to 445.O(O1), SWY(O1) and SWY(O2) in r = 0.84, p < 0.001 in May, r = 0.87, p < 0.001 in June).The dendrogram including all species of molecular compounds detected by FT-ICR MS in May  showed tet al.et al.et al.et al.et al.The results of the FT-ICR MS analysis revealed that thousands of different DOM molecules existed in each water sample collected from our study site, i.e., Kangasvaara catchment . FT-ICR et al.18The linear relationships between DOM molecular diversity and the numbers of lignins, tannins, and CAS  suggest 3629Comparisons of the m/z value between different water samples revealed that common molecular compounds existed at different sampling locations (hypothesis 2). We found that the number of lignins shared between different sampling locations was larger than that of any other biomolecular class. Additionally, correlation analyses between the cophenetic matrices of Jaccard’s distance  showed t4126H32O11 as an example of isomers, which represent two major refractory substances, i.e., lignin and carboxyl rich alicyclic molecule, and explained that these substances have different reactivities and sources though they have the same molecular formula. Therefore, it is possible that even molecular compounds considered lignins in the van Krevelen diagram were misidentified and consequently a larger number of different molecular compounds existed between sampling locations than observed. This also would be supported by Reetsma1230On the other hand, a number of different molecular compounds existed between sampling locations since common molecular compounds accounted for less than 50% of the molecular diversity in the majority of the water samples. This may be related to the fact that the quality of DOM is altered by several processes. For instance, tree canopies and forest soils release DOM molecular compounds into rainwater; whereas in rainwater, they are microbially converted to lower molecular-weight compounds and/or some are retained in mineral soils via co-precipitation40434418 solid phase extraction method used, is incompleteet al.Vertical profiles in DOM molecular diversity and DOC concentration within the Kangasvaara catchment  revealedThis study presents preliminary but unprecedented results on the changes in the molecular diversity and composition of DOM across water movement in a boreal forest. At this stage, we could not examine temporal variations in the quality of DOM. However, common DOM molecular compounds were detected in the same location between May and June and the number of the lignins common to both May and June was larger than that of any other biomolecular class for any water sample . TherefoPicea abies L. Karst.), Scots pine (Pinus sylvestris L.), and white and silver birch . European aspen (Populus tremula L.) were also found in the catchmentThis study was conducted in the Kangasvaara catchment  located O) dominated by Norway spruce and 12-year-old Scots pine plantation (FY). The plantation was established after harvesting a portion of the Norway spruce stands (3 ha–1 in the FO. The field layer vegetation was dominated by dwarf shrubs (Vaccinium vitis-idaea L. and V. myrtillus L.) and the bottom layer by feather mosses (Pleurozium schreberi Brid. and Hylocomium splendens (Hedw.) B. S. & G.). The forest floor consisted of litter and mor humus. In 1996, stem-only harvesting was carried out in 35% of the catchment area and Scots pine seedlings were planted thereafter. Branches, leaves, and treetops, the diameters of which were less than 8 cm, were left on site. The field layer vegetation was dominated by grasses (Deschampsia flexuosa (L.) Trin.) and herbs (Epilobium angustifolium L.), and the bottom layer by pioneer mosses (Dicranum sp. and Polytrichum sp.) in the FY. The average thickness of the overlaying mor humus layer (O-horizon), the eluvial (E) horizon and the upper illuvial (B) horizon was 5.7, 6.8 and 13.9 cm, respectively.The experimental stands in the catchment were old-growth mixed forest (Fe stands . A 50 × 2) in an open area within 500 m of the experimental plots  lysimeters that consisted of 67-mm-long porous cups with an outer diameter of 12 mm . Three lysimeters  were placed 1–2 m apart in every horizon and each stand and soil water from each location was collected for the same week as bulk deposition and throughfall. In May, no water samples were obtained from two lysimeters in the E- and B-horizons in FThe groundwater was obtained from a groundwater tube installed at a depth of 0.7 m in the peat layer (groundwater-PT), 2.5 m in the moraine (groundwater-MO), and 2.4 m in the mineral soil under the peat layer (groundwater-MI). The depth of the peat layer around the groundwater-PT ranged from 1.5 to 2.1 m. The groundwater tube was made of polyamide and had an inner diameter of 30 mm and an outer diameter of 40 mm. The stream water samples were collected at the outlet of the Kangasvaara catchment . The groAll samples were filtered using pre-combusted glass fiber filters with a nominal pore size of 0.7 μm  and frozen until analysis.18 solid phase extraction (SPE) method to remove inorganic salts–1. All samples were analyzed in the negative ion mode. The electrospray voltage was optimized for each sample. Ions were accumulated in a hexapole for 2 s before they were transferred to the ICR cell, and the 100 transients collected using a 2 M Word time domain were co-added. All spectra . DOM molecular compounds were analyzed using electrospray ionization (ESI) coupled to high-resolution FT-ICR MS. In this study, an FT-ICR mass spectrometer with a 9.4-T magnetic field was used . Filtered samples were treated with the C spectra  were extet al.et al.To identify which biomolecular class each molecular compound belonged to, a van Krevelen diagram was used based on the elemental ratios of the expected molecular formulas, i.e., the oxygen-to-carbon (O/C) and hydrogen-to-carbon ratios (H/C)DOC concentration and the number of molecular compounds detected were compared among sampling locations using a one-way ANOVA. The number of replicates ranged from 2 to 4 at each location. Tukey-Kramer post-hoc tests were used to compare sample means. Normality (Shapiro-Wilk test) and homogeneity (Bartlett test) were verified in advance. Jaccard similarity coefficients were calculated to examine how many molecular compounds were the same between different sampling locations, where coefficient = 1 indicates that two water samples share all of the molecular compounds while coefficient = 0 indicates that there are no common molecular compounds. Then, dendrograms of different sampling locations were obtained for all molecular compounds detectable by FT-ICR MS and for each biomolecular class by calculating the Jaccard’s distance between samples and subsequently using Ward’s method. Additionally, correlation coefficients were calculated between cophenetic matrices of Jaccard’s distance for all molecular compounds and for each biomolecular class to examine which biomolecular class dendrogram exhibits a similar pattern to the dendrogram for all molecular compounds. Statistical analyses in this study were conducted using R .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


MR) using phase-contrast (PC)-MRI in conjunction with Navier-Stokes (NS) equations has been attempted in various vessels [2-4]. Our previous work has shown the feasibility of deriving ΔPMR at various vessel diameters in a phantom (fig.1a) and excellent correlation between ΔPMR and ΔP measured via a pressure-transducer (fig.1b). This study aimed to investigate the feasibility of deriving ΔPMR in healthy and diseased coronary arteries.Fractional flow reserve (FFR) is an invasive procedure evaluating the functional significance of an intermediate coronary stenosis in patients with coronary artery disease (CAD) . Quantifo, cardiac phase=2(~70 ms/phase), in-plane resolution = 0.5-0.6 × 0.5-0.6 mm2, slice thickness=3.2 mm and TA=2-4 min/slice at 3T. Eddy-current correction was done offline followed by NS calculations [Coronary PC-MRI acquisitions were ECG triggered (mid-diastole) and navigator gated (end-expiration) . Fat-supp<0.001) increase in ΔPMR was seen in the patient group (6.40 ± 4.43 mmHg) vs. healthy controls (0.62 ± 0.49 mmHg) (figre 2a). CTA/ICA reports in 5/6 patients showed a range of stenoses of 30-50% ), but not significant enough to perform invasive FFR. ICA/FFR was performed in 1/6 patients  with FFR=0.56, suggesting a functionally significant lesion. The same patient showed a ΔPMR of ~14 mmHg, likewise suggesting a functionally significant lesion (relatively high pressure drop).A significant , corroborating the ΔPMR results, showed the feasibility of ΔPMR in differentiating between a functionally significant and a non-significant lesion within the patient group. More patient studies with invasive FFR comparison are underway to further validate the approach. In addition, technical improvements in terms of spatial, temporal resolutions and reduction of noise are also being developed to further improve accuracy.Preliminary results suggest that noninvasive quantification of ΔP


The linear polymer poly, better known by its tradename Kevlar, is an icon of modern materials science due to its remarkable strength, stiffness, and environmental resistance. Here, we propose a new two-dimensional (2D) polymer, “graphamid”, that closely resembles Kevlar in chemical structure, but is mechanically advantaged by virtue of its 2D structure. Using atomistic calculations, we show that graphamid comprises covalently-bonded sheets bridged by a high population of strong intermolecular hydrogen bonds. Molecular and micromechanical calculations predict that these strong intermolecular interactions allow stiff, high strength (6–8 GPa), and tough films from ensembles of finite graphamid molecules. In contrast, traditional 2D materials like graphene have weak intermolecular interactions, leading to ensembles of low strength (0.1–0.5 GPa) and brittle fracture behavior. These results suggest that hydrogen-bonded 2D polymers like graphamid would be transformative in enabling scalable, lightweight, high performance polymer films of unprecedented mechanical performance. Low fracture toughness for these materials persists from monolayer through multilayer6 due to extremely weak inter-layer van der Waals (vdW) interactions, observed in graphite as early as 1928 by Sir William Bragg in his seminal work on crystallography7. Because 2D vdW solids have flaw-sensitivity and low shear strength through all length scales, they have yet to find relevance as stand-alone structural materials. Instead, they are best suited for use as solid lubricants8.Two-dimensional (2D) materials such as graphene and molybdenum disulfide have extraordinary intrinsic stiffness and strength10 provides a new opportunity to design a two-dimensional molecule that forms high strength, hydrogen-bonded solids. Previously proposed 2D polymers have not been designed for inter-molecular hydrogen bonding. Notable examples include covalent organic frameworks (COFs) with hydrazone11 linkages that form intra-molecular hydrogen bonds; or amide-linked, sparse COFs that would likely form a low density of hydrogen bonds12. Additionally, because most COFs are designed with a relatively sparse covalent network, in many cases to achieve tailored porosity for selective chemical separation, their intrinsic molecular strength and stiffness will be lower than denser molecular networks such as graphene.The emergence of 2D polymers13. One particular class of materials, poly , is frequently used in these applications under the tradenames Kevlar and Twaron. These PPTA fibers possess a unique combination of high stiffness and strength with resistance to elevated temperature, chemicals, ultraviolet, moisture, and creep14. This performance is enabled by two key features of the polymer: i) a rigid aromatic backbone, and ii) strong intermolecular hydrogen bonding15. Aromaticity provides stiffness and strength to the molecule, while hydrogen bonding allows for macroscopic strength and stiffness at relatively modest molecular weights16. Intrinsic stiffness and hydrogen bonding also encourages liquid crystalline behavior in solution, leading to spontaneous alignment that simplifies the scaled manufacturing of oriented, chain-extended fibers18.Our new molecule is inspired by commercial high performance polymer fibers, which are among the stiffest and strongest engineering materials per unit mass available todayAnalogous to the highly-aligned, chain-extended linear polymers of PPTA fibers, we propose to create an ensemble of finite molecular weight, aligned, “plane-extended” two-dimensional para-aromatic polyamide molecules, called “graphamid.” Like PPTA solids, strong hydrogen bonding between graphamid molecules generate high ensemble strength and stiffness at molecular weights that should be achievable via scalable polymer synthesis techniques. Unlike PPTA, however, we will show that all individual sheets in graphamid are bonded either covalently or by hydrogen bonds, leading to superior predicted strength and stiffness. Furthermore, the 2D nature of the graphamid bond network gives it strength and stiffness in all in-plane directions, unlike the unidirectional properties of oriented linear PPTA. Finally, we demonstrate that hydrogen bonding in graphamid leads to superior fracture toughness compared to vdW solids like graphite.6 aromatic rings interconnected in six directions by amide bridges to form a 2D covalent polymer. In contrast, PPTA, the inspiration for graphamid, has aromatic rings linked in two opposing directions by amide bridges, forming long linear polymeric chains  were used to predict the lowest energy configuration of graphamid, as shown by the 72 atom rectangular primitive cell Fig. . Graphamins Fig. .Figure 119  versus engineering strain (ε) responses of the four materials  is higher than the values for graphamid. For uniaxial loading applications, such as ropes and cables, linear PPTA oriented along the 1NN direction would be a more ideal molecular structure than graphamid. However, for applications in which multi-axial loading is expected, such as membrane and barrier applications, isotropically averaged properties are more relevant. The PPTA crystallite arranges into hydrogen bonded “sheets”, which are themselves bonded via weaker van der Waals bondsThe behavior of molecular ensembles will be strongly dependent on inter-molecular shear behavior. To determine these properties, bilayer molecular stacks were simulated in DFT for graphene, graphylene, and graphamid. System energies were calculated at various layer-to-layer relative displacements, with the lowest energy bilayer configurations shown in Fig. 3) is significantly higher than that of graphylene (1.49 g/cm3) due primarily to the presence of oxygen in the graphamid structure. Crystalline spacing in PPTA were also calculated  of 960 GPa and a strength of 120 GPa, consistent with prior computational21 and experimental23 studies. Graphamid has a stiffness of 260 GPa and a strength of 28 GPa, compared to 208 GPa and 33 GPa for graphylene. PPTA is predicted to have an intrinsic molecular stiffness and strength along the chain direction of 334 GPa and 32 GPa, respectively, which is in reasonable agreement with prior estimations for the PPTA crystallite from theory and experiments  at a finite distance from the end of the molecule. Because molecules are not being fully and uniformly loaded along their length, the global average elastic response is less stiff than the intrinsic molecular stiffness. This shear lag effect also produces intermolecular shear stresses that are maximized at the molecule ends. Ensemble failure is predicted to occur when the local shear stress at a molecular end point exceeds the intermolecular shear strength of the material. A detailed discussion of these theories, and validation of the predictions as applied to PPTA fibers, can be found in Northolt et al.25.To predict the global mechanical behavior of ensembles of finite sized molecules, we follow the approach of YoonEens and strength σens,c, under conditions of perfect orientation, are:We can expand the Yoon theory, originally derived for linear molecular ensembles, to predict the behavior of ensembles of platelet-like 2D molecules . The resulting predictions for ensemble film elastic modulus E and G are the polymer Young’s and inter-molecular shear modulii respectively, τc is the intermolecular shear strength, p = L/a is the aspect ratio of the molecule, L is molecular length, a is intermolecular distance, and α = β = 1 are pre-factors associated with the platelet geometry. The molecular cohesive parameter, δ, accounts for the fact that the fundamental molecular domain may not be a single molecule (δ = 1), but may in fact be an associated group of molecules that behave as a cohesive unit without internal shear. This molecular domain has an effective aspect ratio of p/δ. In oriented polymer fibers, this effect is observed as 1–100 nm diameter “fibrils” that emerge during fiber deformation and failure. Following Northolt et al.25, we assume the domains consist of ten cohesive molecules (δ = 10) for the present analysis. The precise value selected for δ determines a shift factor for material properties relative to absolute molecular weight, but does not have a significant effect on the comparative behavior of graphamid relative to the benchmark materials of the present study  for these materials. Hydrogen bonding in graphamid reduces shear lag, increasing molecular loading at lower aspect ratios  compared to graphene and graphylene. Strength predictions show that graphamid is expected to provide the highest strength for molecular ensembles, with values above 10 GPa possible for molecular lengths above 200 nm. Graphene and graphylene show significantly lower asymptotic strengths, due to their lower intermolecular shear strength values, and require higher molecular weights due their low shear modulus.Using equations  and 3),,3), and ons Fig. , predict26, we estimate the in-plane modulus Eens(θ) of an ensemble film with uniform molecular orientation θ relative to the film plane, asEens is the film modulus for a perfectly oriented, finite molecular weight ensemble from Eqn.  asThe modulus and strength predictions in Eqns  and 3)  3) assummentclass2pt{minimθ = 10°, within the range of θ = 5°−16° measured for commercial PPTA fibers of various grades28. Due to the very low shear modulus of graphene and graphylene, at this inclination angle graphamid exhibits the highest predicted stiffness values, reaching an asymptotic value of 126 GPa compared to 105 and 70 GPa for graphene and graphylene, respectively. For strength values, the very low shear strength of graphene and graphylene drive their mechanical strength values to less than 2 GPa, while graphamid is predicted to exceed 8 GPa strength at molecular lengths as short as 100 nm. These predictions show that the presence of hydrogen bonding in graphamid provides significant benefits in terms of strength and stiffness in molecular ensembles, and suggests that molecular weights of a few hundred nanometers should be sufficient to achieve films of outstanding strength and stiffness.Figure 29) relative to our theoretical strength predictions (1–2 GPa), are suggestive that poor orientation, wrinkles, lattice defects, or other factors have significantly limited the performance of these materials. A comparison of graphamid relative to graphene oxide (GO) papers and composites, functionalized for enhanced intermolecular bonding, is provided in a later discussion.Graphene papers are ensemble membranes that have been synthesized, but have relatively low strength compared to graphamid due to their lack of hydrogen bonding . In fact, the low strength of graphene papers . Figure E , shear parameters from Fig. δ = 10. The stiffness and strength ranges predicted for PPTA are comparable to experimental values for commercial PPTA fibers with average molecular length above 100 nm28, providing confidence in the theory and calculations  can  can 5) cWe performed atomistic fracture simulations for multilayer domains of graphene, graphylene and graphamid to demonstrate that inter-layer attraction leads to a fundamental increase in fracture toughness for graphamid relative to vdW solids. Classical molecular dynamics (MD) using the ReaxFF potential (see Methods section) was used to simulate uniaxial tension on graphene, graphylene and graphamid in the following configurations: i) a monolayer (1L) with a mode-I crack, and ii) a three layer (3L) stack with a mode-I crack in the middle layer Fig. . MD predUfracture, 44% larger than that of graphene, and 124% higher than that of graphylene . When examining 3L fracture, not only does graphamid have the highest fracture stress, it has an energy to fracture 1.8× higher than graphene and 2.8× higher than graphylene. While the 1L fracture energy of graphamid is the highest of the three materials, the 3L fracture energy of graphamid is disproportionately higher than 3L graphene and graphylene due to multilayer enhancement from hydrogen bonding. This enhancement is best seen by the increase in strain to failure of graphamid which goes from a value of ε = 8.1% in 1L to ε = 11.8% in 3L, meanwhile graphene and graphylene fail at nearly the same strain in the 1L and 3L configurations, around ε = 4% and ε = 5% respectively. The results for comparison of stress, strain and energy to fracture for the different scenarios is summarized in Supplemental Table The stress-strain response for pre-cracked 3L domains are compared to 1L domains with the same area and pre-crack length for graphene, graphylene and graphamid in Fig. KIC, was calculated for the 1L domains, as well as in the middle layer only for the 3L domains. The middle layer in the 3L fracture configuration was examined for stress concentration because the outer two layers are pristine, and this extrinsic stress concentration factor can provide a quantification for the reduction in stress localization due to load-sharing between layers. Local y-component stress, σyy, is plotted as a function of distance from the crack tip, r, from the crack to the edge of the domain as seen in Fig. Jyy,, in rectangular bins, and the resulting stress profile is used to calculate KIC according to the following equation:σfar is the far-field stress. The reduction of stress concentration in 3L graphamid compared to 1L can be seen in the virial stress energy plotted in Fig. ε = 6.9% has a distinct stress concentration at the crack tip  papers. Thus far, this approach has resulted in only modest strength achievements, reaching values of 0.03–0.3 GPa2, only a 13% lower hydrogen bond population than graphamid. In this scenario, the maximum shear strength in multilayer GO can be estimated from the hydrogen bond energy (0.32 eV)31, the bond distance 31, and the ideal bond density (7 bonds/nm2), yielding a value of τc,Max = 1.05 GPa . Given the approximate nature of this estimation, it is reasonable to state that such a shear strength value is comparable to our predictions for graphamid (1.45 GPa).To maximize the strength of an intermolecular interface in a GO paper, hyrdoxyl and epoxide groups on one sheet would be spatially matched with complementary epoxide and hydroxyl groups, respectively, on the adjacent sheet. Theoretically such hydrogen bonds could be packed at a density of 7 bonds/nm32. Through modified growth methods it was found that, due to steric exclusion, the maximum functionalization density is approximately 85%33. Secondly, achieving theoretical shear strengths in GO would require perfect complementary patterning of hydroxyl and epoxide groups on opposing GO faces. In reality, these functional groups populate randomly on each surface, drastically reducing the likelihood of hydrogen bond formation. In addition, GO has limited inter-layer shear strength due to self-passivation of hydrogen bonds within a given layer. One simulation study found that in multilayer GO, nearly half of hydrogen bonds are intra-layer bonds that do not contribute to shear strength, a result that held true even when the simulations were repeated with a reduced population of functionalization31. Finally, epoxides cause significant and random corrugation in GO, diminishing contact area between layers and further diminishing the likelihood of hydrogen bond formation. The combined effect of all of these factors results in a true hydrogen bond density at least 10× lower than the theoretical maximum , and a commensurate order-of-magnitude loss in shear strength, leading to the poor mechanical properties observed experimentally for GO ensembles.Unfortunately, however, experimentally realizing such theoretical intermolecular bonding densities is highly unlikely. First, GO papers are created by oxidation of graphene sheets, and experimental functionalization densities  are typically 20–50%, with the highest reported density at 70%34. Secondly, the graphamid molecule possesses these amide groups at high per-area density, and the equilibrium configuration of graphamid is effectively planar with the amide groups rotated out of plane. This molecular structure results in a high density of accessible intermolecular hydrogen bonds that would be expected to approach our theoretical predictions. Analogous advantages in PPTA - a high population of self-aligning amide groups in a “rigid rod” polymer - explain why PPTA forms high strength fibers, and why our theoretical shear strength predictions from DFT agree well with the limited experimental data available.In contrast, graphamid has a number of fundamental advantages. First, the amide group forms hydrogen bonds with other amide groups. Therefore, there is no requirement to create matched complementary functional group patterns as is necessary with GO. Instead, simply creating self-similar graphamid molecules of good crystallinity will result in dense hydrogen bonding. For this reason, and due to the synthetic accessibility of amide chemistries, it is a preferred functional group for creating hydrogen bonded polymersintra-molecular hydrogen bonds, with the amide groups rotated into the molecular plane12. It is apparent that the tight packing of the graphamid structure forces the amide groups to rotate into a state that is amenable to intermolecular hydrogen bonding. The combination of short amide groups bridging nodal benzene rings to form a 6-fold planar covalent bond network could make graphamid an archetypal molecule for creating compact, stiff, and strong hydrogen-bonded 2D molecular ensembles.For these same fundamental reasons, the graphamid structure is likely to be one of the most ideal 2D polymers for creating hydrogen-bonded ensembles. For comparison, amide-containing sparse covalent organic frameworks generate 35. The same factors that limit strength development in GO papers, namely low bond density, are also likely concerns for these composites. We also note that polymer loadings in these systems are typically around 50%. In contrast, a graphamid solid does not require an intermediary polymer that would reduce the per-mass efficiency of the film, i.e. graphamid is a “100% volume fraction” reinforced material.An approach related to GO papers is to build polymer-GO composites, using a low molecular weight polymer to mediate hydrogen bonds between layers, or form direct physical crosslinks. These materials have demonstrated good toughness, but strengths remain below 0.5 GPa3, a disruptive improvement over typical high performance biaxially oriented films, or quasi-isotropic stacks of woven PPTA20. In fact, the strong hydrogen bonding in graphamid suggests that it will be resistant to both in-plane tension and compression, making it suitable for a wide range of structural applications. In contast, the weak van der Waals planes in PPTA fibers lead to kink band failures at low longitudinal compression loads36, restricting the PPTA market primarily to tensile applications such as ropes and body armor. In addition, the high stiffness of graphamid and high intermolecular interactions make it likely that it will form liquid crystalline solutions, an important characteristic for enabling scalable oriented film production. Considering the revolution in high performance materials triggered by the discovery and commercialization of Kevlar 50 years ago, and given the rapid, recent progress in COF and 2D polymer synthesis, we are hopeful that the promise of materials like graphamid generates the rapid investment and discovery necessary for a 2D polymers revolution in the 21st century.Compared to vdW solids like stacked graphene, the present analysis shows that hydrogen-bonded 2D polymers would be expected to exhibit dramatic advantages for creating films of extraordinarily high strength, stiffness, and toughness. Hydrogen bonding provides a number of key advantages. First, commercial synthesis of infinite molecular sheets of 2D polymer is likely to be exceedingly difficult. Instead, materials like graphamid will most likely be industrialized in a manner comparable to linear polymers, i.e. solution synthesis of countless finite-sized, sub-micron molecules that will then be deposited into an oriented film. The ensemble mechanical analysis demonstrates that strong intermolecular hydrogen bonding is key to achieving high strength and stiffness in such molecular ensembles. These effects become even more important as molecular orientations deviate from perfect alignment, another reality of scaled manufacturing. While this work does not consider all manufacturing-induced imperfections that can impact mechanical performance, such as contaminants, the results of neat graphamid compared to neat PPTA indicate that graphamid will be a superior mechanical material under comparable flaw conditions. Second, hydrogen-bonding between layers of graphamid provide a toughening mechanism during fracture wherein local loads are transferred to neighboring layers, diminishing stress concentration around a crack defect. Therefore, unlike vdW solids, graphamid solids should be flaw tolerant and extremely tough. Quantitatively, ensemble theory predicts that graphamid films composed with likely molecular lengths and orientations will result in films with isotropic in-plane stiffness and strength of 110 GPa and 6 GPa, and a density of 1.8 g/cm37 in conjunction with the widely used generalized gradient approximation exchange-correlation functional of Perdew, Burke, and Ernzerhof (PBE)38. The PBE functional was compared to and chosen over the Becke, Lee, Yang, and Parr40 (BLYP) functional due to higher accuracy in reproducing PPTA lattice constants with respect to the experiment25. Using the computational setup described below and a simulation cell of 336 atoms with dimensions ~15 × 15 × 13 Å, we find that the lattice parameters are most closely matched to experimental data for the PBE functional  and a multi-grid plane wave basis for the electron density. Goedecker, Teter, and Hutter (GTH)44 pseudopotentials are used to treat the cores for carbon, nitrogen, oxygen and hydrogen. These separable dual-space Gaussian-type pseudopotentials are optimized for PBE by Krack45. A Gaussian basis set with triple-zeta with valence polarization functions is used for the single body orbitals, as is a plane wave cutoff of 350 Ry for the electron density. Energy self-consistence was converged to 10−6 Ha, forces were converged to 4.5 × 10−4 Ha/bohr, and stress tensor elements were converged to below 100 bar. While the structures we study are expected to be nearly charge balanced we still employ a dipole correction as described by Bengtsson46 to treat any serendipitous polarization due to lack of rigorous symmetry. Dispersion interactions are accounted for using a 1.5 nm cutoff with the Grimme DFT-D method with D3 parameterization as implemented in CP2K48. Results are given for the gamma point where the simulation cell contains multiples of 72 atoms depending on the number of layers studied. All first-principles calculations use periodic boundary conditions. The simulation cell lattice vectors are more than 1 nm in length and nearly orthogonal; the out of graphamid plane lattice vector was no less than 3 nm in length.We performed our DFT calculations using the freely available CP2K code6 graphamid rings correspond to graphene atom sites and the graphamid –CO–NH– chains correspond to graphene bonds.In addition to initial zero pressure graphamid structures we also computed uniaxial stress-strain curves via DFT. We treat the strain directions similar to graphene. Graphamid is analogous to graphene if the strong C−1, similar to previous publications19 . Simulations of GrE-2 were performed in AIREBO49 with a time step of 0.5 fs and a carbon-carbon interaction cutoff of 0.197 nm to avoid aphysical strain hardening, in accordance with prior literature as well as previous DFT validation for 2D polymer systems19. The unmodified ReaxFF potential50 was used for graphamid simulations, with a time step of 0.2 fs. Local 2D stress is determined by binning atomic virial stress energy and area-averaging the values. Atomic virial stress energy maps are performed by linearly interpolating the binned energy values.Classical MD simulations were equilibrated at 0 K and subsequently integrated under constant energy (NVE) with a strain rate of 2.0 nsSupplementary Information


Molecular estimates of phylogenetic relationships rely heavily on multiple sequence alignment construction. There has been little consensus, however, on how to properly address issues pertaining to the alignment of variable regions. Here, we construct alignments from four commonly sequenced molecular markers  for the Nudibranchia using three different methodologies: (i) strict mathematical algorithm; (ii) exclusion of variable or divergent regions and (iii) manually curated, and examine how different alignment construction methods can affect phylogenetic signal and phylogenetic estimates for the suborder Doridina. Phylogenetic informativeness (PI) profiles suggest that the molecular markers tested lack the power to resolve relationships at the base of the Doridina, while being more robust at family-level classifications. This supports the lack of consistent resolution between the 19 families within the Doridina across all three alignments. Most of the 19 families were recovered as monophyletic, and instances of non-monophyletic families were consistently recovered between analyses. We conclude that the alignment of variable regions has some effect on phylogenetic estimates of the Doridina, but these effects can vary depending on the size and scope of the phylogenetic query and PI of molecular markers. The wrongful designation of characters as homologous can confound interpretations of evolutionary relationships by creating homoplasies, which results in the loss of phylogenetic signal . Thus, tHighly divergent sequences and a general lack of diverse molecular markers have made MSAs and resulting phylogenetic estimates for the Nudibranchia problematic –26. For The Doridina suborder currently consists of 19 families and more than 2000 described species. The classification has been divided into five superfamilies  defined by morphological variation in gill and feeding structures ,36,58,59In this study, we use three common MSA methodologies: (i) strict mathematical algorithm; (ii) exclusion of variable or divergent regions and (iii) manually curated. We then discuss issues pertaining to the lack of resolution in phylogenetic estimates and limitations in resolving the base of the Doridina. We also address the possible effects alignment construction may have on phylogenetic informativeness (PI), and how phylogenetic signal may shift given alignment methodology. Lastly, we re-evaluate taxonomic classifications that were consistent between all three analyses.2.2.1.Pleurobranchaea meckeli and Berthella martensi were chosen as outgroups because the Pleurobranchomorpha has been suggested to be sister to the Nudibranchia ).Even though the focus of the present study was to understand MSA construction, our estimates of the Doridina also give a frame of reference for allowing more intensive queries into specific family evolutions. For example, the evolution of caryophyllidia in the Discodorididae, molecular evolution in Aegiridae and Dendrodorididae, or the relationships pertaining to the Polyceridae, Okakaiidae, Gymnodorididae, Hexabranchidae and Chromodorididae, which are some of the most morphologically unique and chemically distinct families.Nudibranch studies unquestionably suffer from a lack of abundant and diverse molecular markers. Studies have argued that increasing molecular markers could resolve problematic relationships ,102, but


We used data from the Global Burden of Disease 2015 study (GBD) to calculate the burden of lower respiratory infections (LRIs) in the 22 countries of the Eastern Mediterranean Region (EMR) from 1990 to 2015.Haemophilus influenzae type b, Respiratory syncytial virus, and influenza virus. We used modeling methods to estimate incidence, deaths, and disability-adjusted life-years (DALYs). We calculated burden attributable to known risk factors for LRI.We conducted a systematic analysis of mortality and morbidity data for LRI and its specific etiologic factors, including pneumococcus, In 2015, LRIs were the fourth-leading cause of DALYs, causing 11,098,243  DALYs and 191,114  deaths. The LRI DALY rates were higher than global estimates in 2015. The highest and lowest age-standardized rates of DALYs were observed in Somalia and Lebanon, respectively. Undernutrition in childhood and ambient particulate matter air pollution in the elderly were the main risk factors.Our findings call for public health strategies to reduce the level of risk factors in each age group, especially vulnerable child and elderly populations.The online version of this article (doi:10.1007/s00038-017-1007-0) contains supplementary material, which is available to authorized users. Lower respiratory infections (LRIs) are one of the leading causes of death and disability-adjusted life-years (DALYs) worldwide, especially among children under 5  and Haemophilus influenzae type b (Hib) in children and high-risk populations in EMRs, and improved access to antibiotics and supportive care have decreased the incidence and fatality of LRI in many countries  per capita. The low-income countries (LICs) are Afghanistan, Djibouti, Somalia, and Yemen. The middle-income countries (MICs) are Egypt, Iran, Iraq, Jordan, Lebanon, Libya, Morocco, Pakistan, Palestine, Sudan, Syria, and Tunisia. The high-income countries (HICs) are Bahrain, Kuwait, Oman, Qatar, Saudi Arabia, and the United Arab Emirates (UAE).All types of LRI  were included in this study. We used the following International Classification of Diseases (ICD-10) codes (or their corresponding codes from earlier ICD versions) as equivalent to LRI: J09–J15.8, J16–J16.9, J20–J21.9, P23-P23.9, and Z25.1. The ICD-10 codes for etiologic categories of LRI included J09–J11.89 and Z25.1 for Influenza virus, J12.1 for RSV, J13, J13.0, J15.3, J15.4, and J15.6 for pneumococcus, J14 and J14.0 for Hib, and J12, J12.0, J12.2–J12.9, J15–J15.9, J15.5, J15.7, J15.8, J16–J16.9, J20–J21.9, and P23-P23.9 for other LRI. We did not include tuberculosis in this study; it has been classified as a separate item in GBD.An analysis of available data on all-cause mortality for all countries was undertaken  which contained data on LRI. A list of all data sources is available on the Institute for Health Metrics and Evaluation’s website.  A series of Bayesian meta-regression analyses through DisMod-MR 2.1 were used for disease modeling. Model-based epidemiological estimates in combination with disability weights were used to calculate cause-specific YLDs for each age, sex, location, and calendar year. DALYs were calculated through summation of YLLs and YLDs , non-exclusive breastfeeding, zinc deficiency, smoking, secondhand smoke exposure, household air pollution from solid fuels, and ambient air pollution as known LRI risk factors. Details on definitions and measures of age- and sex-specific relative risks of LRI for each of the risk factors are available elsewhere  for each estimate—such as rates or numbers of deaths or DALYs. We estimated UIs by taking 1000 samples from the posterior distribution of each quantity and using the 25th- and 975th-ordered draws of the uncertainty distribution.LRI deaths decreased by 30.2% from 273,714  in 1990 to 191,114  in 2015 in EMR countries. In spite of the decrease, LRI is still the third-leading cause of death for all ages in 2015. The death rate per 100,000 was 74.0 (95% UI 64.8–82.9) in 1990 and decreased to 29.5 (95% UI 26.4–32.5) in 2015, a 60.1% reduction. The age-standardized death rate for LRI was 65.4 (95% UI 58.6–73.4) in 1990 and declined by 38.5% to 40.2 (95% UI 36.0–45.2) in 2015. In 2015, 4.8% (95% UI 4.3–5.2) of all deaths and 13.0% (95% UI 11.5–14.7) of under-5 deaths were due to LRI.At the regional level, the highest death rates and numbers of deaths were among children under 5 years old, followed by those aged 65 years or older. However, there was considerable heterogeneity in the age pattern of LRI deaths between the countries of the EMR. In all high-income countries , and some of the middle-income countries , the LRI death rates among older age groups were greater than the mortality rates among children under 5 years. Death rates were 31.5 per 100,000 (95% UI 27.3–35.4) in men compared to 27.4 per 100,000 in women (95% UI 23.9–31.2) in the region. The LRI death rate was not different in EMR countries in boys versus girls under 5 (2015).Figure Haemophilus influenzae type b , and influenza virus . Figure Among the etiologic causes, pneumococcus had the highest mortality rate , followed by respiratory syncytial virus , The rate of YLLs per 100,000 population decreased by 69.5% from 5579  in 1990–1702  in 2015. However, LRI was still the third-leading cause of YLLs in 2015.e-Table 1 shows age-standardized incidence rates of LRI in males and females of the EMR countries. The highest incidence rate was observed in Afghanistan and the lowest in Tunisia for both sexes. In all countries but Afghanistan, Bahrain, and Djibouti, the incidence rates were higher among men than women, with the highest male-to-female ratio in Iran and Libya.The rate of YLDs per 100,000 decreased from 15.8 (95% UI 10.7–22.9) to 9.7 (95% UI 6.5–13.6) per 100,000 during 1990–2015.Total DALYs from LRI decreased by 40.6%, from 20,746,747  in 1990 to 11,098,243  in 2015. LRI, which was the leading cause of DALYs in 1990, was ranked fourth in 2015, behind ischemic heart disease, neonatal preterm birth complications, and neonatal encephalopathy. LRI DALY rates were 5594 (95% UI 4851–6334) and 1712 (95% UI 1520–1912) DALYs per 100,000 population in 1990 and 2015, respectively, a 69.4% reduction. About 4.9% (95% UI 4.3–5.5) of DALYs in all ages and 12.2% (95% UI 10.8–13.8) of DALYs in children under 5 were due to LRI in 2015.Age-standardized DALY rates were 3411 per 100,000  in 1990 compared to 1518 per 100,000 (95% UI 1357–1673) in 2015 in the EMR. Global age-standardized DALY rates for LRI were lower than those in the EMR: 3310 (95% UI 3033–3551) in 1990, and 1428 (95% UI 1330–1511) in 2013. There was a 56.9% reduction in age-standardized DALY rates at the global level, which was similar to the EMR (55.5% reduction) during 1990–2015. Figure Figure Figure Figure Our study showed a substantial burden of LRI in the EMR. Despite a decline in the burden of LRI since 1990, LRI remained the fourth-leading cause of DALYs and third-leading cause of death in the region in 2015. The all-ages burden of LRI in the EMR is considerably higher than global rates, and even the age-standardized burden is slightly higher. Our findings call for intensified efforts to control and reduce the burden of LRI in the EMR, especially among low-income countries and low-resource settings. Moreover, since the current situation of health status and LRI varies widely between the EMR countries, suggested interventions to avert LRI deaths and disabilities should be specified at the national or even subnational level  to their national immunization programs. Moreover, all low-income countries in the region except Somalia have introduced PCV thanks to support from Gavi, the Vaccine Alliance. Gavi .All EMR countries have introduced Hib vaccine to their immunization programs, and coverage of Hib3 in the EMR has increased from 13% in 2005 to 72% in 2014. However, coverage of Hib3 varies across EMR countries  are important therapeutic interventions for LRI control , and MERS-CoV Supplementary material 2 (XLSX 25 kb)Below is the link to the electronic supplementary material.


Public health messaging about antimicrobial resistance (AMR) sometimes conveys the problem as an epidemic. We outline why AMR is a serious endemic problem manifested in hospital and community-acquired infections.AMR is not an epidemic condition, but may complicate epidemics, which are characterised by sudden societal impact due to rapid rise in cases over a short timescale. Influenza, which causes direct viral effects, or secondary bacterial complications is the most likely cause of an epidemic or pandemic where AMR may be a problem. We discuss other possible causes of a pandemic with AMR, and present a risk assessment formula to estimate the impact of AMR during a pandemic. Finally, we flag the potential impact of genetic engineering of pathogens on global risk and how this could radically change the epidemiology of AMR as we know it.Understanding the epidemiology of AMR is key to successfully addressing the problem. AMR is an endemic condition but can play a role in epidemics or pandemics, and we present a risk analysis method for assessing the impact of AMR in a pandemic. Antimicrobial resistance (AMR) is the resistance of bacteria, viruses or other micro-organisms to antimicrobial drugs. The most common and concerning form of AMR is antibiotic resistance against important bacteria. Many multi-resistant bacteria have emerged over the past century. AMR is driven by selective evolutionary pressure, and relates to the volume of antibiotic use through over-use or misuse in humans, animals and food production. AMR can also occur through random genetic mutations. Efforts to control antibiotic use have focused on human and animal health. AMR is an increasing concern globally, and can increase the overall impact of any infection by reducing or removing the ability to treat infections successfully. In this scenario the rate of illness and death would be the same as that seen in untreated infection.0. A R0 of 1 is the epidemic threshold, and any R value greater than 1 presents necessary conditions for an epidemic. The rapid increase in cases observed during an epidemic or pandemic is what causes immediate impacts on health systems, stressing these systems beyond normal operating capacity. A pandemic of a highly infectious microbe will evolve rapidly and affect many people within a short period of time. For example, an influenza pandemic once it begins, would be expected to reach its peak within 2 months. A working group commissioned by the World Health Organization (WHO) has listed diseases with pandemic potential, but some of these, such as malaria, TB and AMR organisms, are usually endemic diseases [Epidemic and pandemic diseases must be differentiated from endemic diseases, such as malaria and tuberculosis (TB), which can exist in high numbers in a population and cause high burden of disease, but with a constant rate or slow change in rates. A true epidemic has a rise in case rates over days or weeks, whereas an endemic disease may rise or fall over years or decades. The words epidemic and pandemic are often misused. A pandemic is simply a global epidemic, and an epidemic is an outbreak of disease that attacks many people at about the same time and spreads through a defined population. It is defined by rate of growth of the epidemic curve. Epidemics have a sudden and immediate impact on the health system and require surge capacity. For transmissible diseases, epidemics have a mathematical definition related to the reproductive number, RStaphylococcus aureus (MRSA) and multidrug resistant TB are well known examples, but increasingly common causes of urine infection and pneumonia are also becoming resistant. AMR is not an epidemic, but an endemic condition.AMR has been a chronic, slowly increasing phenomenon worldwide, and causes long term impact on the health system, as well as severe, immediate impact on affected individuals. Hospital infections such as Methicillin-resistant However, AMR can complicate an epidemic or pandemic. In the case of a pandemic, where large scale infections would affect the population in a short space of time, AMR could contribute adversely to serious illness and death on a mass scale. The mortality impact of AMR on a pandemic of any infection depends on a series of factors. Whilst there are other contributors, the main determinants are whether or not there is a drug available to treat the infection. If this is zero, then the impact of AMR is zero. It also depends on: the degree of AMR – whether it is partial, or complete; the total mortality caused by the infection; and the proportion of deaths preventable by that drug. These factors together, determine the impact of AMR on a pandemic. The equation can also be modified for morbidity. Many emerging infections (such as Ebola and MERS) have no available drug for treatment, so the impact of AMR is zero. The overall mortality impact of AMR can be summarised by Fig. Influenza is the most likely cause of a pandemic where AMR would be a factor. Influenza morbidity and mortality can be due to the virus itself or to secondary bacterial infections, therefore AMR can doubly impact an influenza pandemic through both antiviral and antibiotic resistance. Based on data from the 2009 influenza A(H1N1) pandemic, the case fatality rate could be anything from 1 to 20% in the absence of substantial AMR . BacteriAMR may be primary or secondary. Primary AMR occurs when the infecting microbe is resistant at the outset and secondary AMR is when resistance develops during treatment, which may occur when people do not finish a full course of antibiotics or when antibiotics are used inappropriately. AMR infections may arise in a surgical wound, at the site of an intravenous cannula, or other skin surfaces which have been breached due to a medical procedure. Resistant infections can also arise in the bowel, urine or the lungs  and are a risk in severely ill patients in intensive care. The impact of hospital acquired AMR is serious for the individual patient and can result in severe medical complications, extended hospital length of stay and death. Hospital acquired infections contribute significantly to avoidable illness and death. In the United States (US) over 2 million people acquire an infection from a hospital each year, with around 70% of these hospital-acquired infections caused by AMR strains . Those wA 2014 review paper on AMR  estimateAMR may cause acute burden of disease in a population during a pandemic, depending on various factors. These include the ability of the microbe to spread rapidly and cause a pandemic or large epidemic ; the severity of the disease caused by the microbe; the degree of AMR ; and the number of drugs to which resistance is present – multidrug resistant strains have a greater impact. In addition, whether there are alternative drugs to use in place of the ineffective drug and the extent which disease control efforts rely on the use of antimicrobials to mitigate a pandemic will also have an impact.0, incubation period and transmission rates. The burden of AMR during a pandemic will also vary for different regions – for example, low income regions will likely suffer a higher burden due to poorer health care standards in hospitals, greater crowding, limited access to laboratories, widespread purchasing of antimicrobials without prescription and poor regulatory frameworks for antibiotic use.These factors are influenced by inherent characteristics of the pathogens themselves in addition to external contextual factors. For example, the ability of the microbe to spread is determined by epidemiological parameters including ROf the diseases that have potential to cause a pandemic where AMR may be a contributing factor to health impact, influenza is the most likely. Influenza has potential to cause large scale morbidity and mortality over a relatively short period of time due to rapid spread between people, and there are commercially available antiviral drugs currently available for use in the event of an influenza pandemic. Antivirals will be heavily relied upon for control of a pandemic in the early phase, as the development of a vaccine matched to the pandemic influenza strain will take up to 6 months. In 2003, adamantane-resistant H3N2 influenza strains emerged, rendering adamantanes ineffective for treatment. The main class of antivirals available presently are the neuraminidase inhibitors, including oseltamivir and zanamivir. Dual adamantane-oseltamivir resistant strains have been reported sporadically in seasonal H1N1 in Cambodia (2007) and Hong Kong (2008) but are not considered to be widespread.In 2007 oseltamivir-resistant seasonal influenza H1N1 (A/Brisbane/59/07) strains emerged as primary resistance in Europe. Unlike other anti-viral resistant strains of influenza, this particular virus strain was able to transmit efficiently from person to person. Within a year, the strain had spread globally, including in regions where oseltamivir was not used. Following the emergence of the H1N1pdm09 virus in 2009, the oseltamivir-resistant seasonal influenza H1N1 (A/Brisbane/59/07) strains died out, and strains circulating across the globe since then have been largely sensitive to NAIs. The fact that only a small number of oseltamivir resistance H1N1pdm09 viruses have been found so far, suggests that resistant strains may have reduced ability to transmit from person to person, and hence may not interfere with the efficacy of high levels of antiviral usage. The largest documented community outbreak of oseltamivir-resistant H1N1pdm09 strains was identified in Australia in 2011, and involved only 29 people [Streptococcus pneumoniae – the percentage of bacteria resistant to penicillin varies widely according to resistance definitions  and regne study , and up ne study . There aStaphylococcus aureus and Haemophilus influenzae. AMR is a significant concern as community-acquired MRSA have been reported to cause complications in seasonal influenza seasons and during the 2009 pandemic. In a sample of 838 critically ill minors during the 2009 influenza pandemic in the US, 33% had evidence of bacterial confection, of these, 39% were attributed to Staphylococcus aureus, and among these 58% were methicillin-resistant [Bacterial coinfections have been reported to play a role in around a third of influenza cases managed in ICUs and up to 30–50% of fatalities are attributed to bacterial infections . Other besistant . During esistant .Enterococcus (VRE), Pseudomonas, Acinetobacter and drug resistant pneumococci, and (ii) those which cause intrinsic AMR such as Clostridium difficile.Other multi-drug resistant bacteria with the potential to impact morbidity and mortality during an influenza pandemic especially in ventilated ICU patients include (i) those which cause acquired AMR such as vancomycin resistant Yersinia pestis which often infects small rodents and is transmitted to humans through the bite of an infected flea. However this disease is unlikely to occur in high income settings which have good sanitation. Historical pandemics caused by the plague include the ‘the Justinian Plague’ of 541 AD, the ‘Black Death’ which emerged in China in 1334, and the ‘Modern Plague’ which began in China in the 1860s. Large scale epidemics of plague are unlikely to occur in countries where there are no animal reservoirs for Y. pestis. Whilst plague is an unlikely cause of a pandemic, it can be treated with antibiotics, therefore AMR would substantially increase the fatality rate of the disease.Past pandemics have been caused by the bubonic plague, caused by the bacterium Smallpox is caused by the variola virus and was globally eradicated in 1980 following a vaccination campaign led by the WHO. Small pox is contagious and spreads through respiratory droplets from infected cases as well as contact. Smallpox is only transmissible while patients are symptomatic. It does not usually cause very large outbreaks and spread usually occurs to members living in the same household. Large outbreaks in schools were not commonly reported. Fatalities occur in up to 30% of cases . The smallpox virus officially exists in just two high-security laboratories in Russia and the US for scientific research, hence there is still a risk of the smallpox virus escaping the laboratory and causing a pandemic. The genome of smallpox is also fully sequenced and known, so it is technically possible for smallpox to be synthetically engineered in a laboratory. Many governments procure and stockpile antivirals for use in the event of a smallpox outbreak – hence the development of AMR may be relevant in the event of a smallpox pandemic. However, little is known about the effectiveness of currently available antivirals such as cidofivir and tecovirimat. It is assumed they may be effective based on lab experiments, however, because these drugs were not tested in people with smallpox, it is not known how effective they will be. No antiviral resistant smallpox strains have been reported so far. Secondary bacterial infections of the skin can also occur as a complication of smallpox, and would be treated by penicillin and cephalosporin antibiotics. These would be more likely to be affected by AMR, but the net impact on morbidity and mortality is unknown. The equation provided above could be applied to a smallpox outbreak at the time, as real data becomes available, to estimate impact.In their natural state, diseases such as TB, malaria and HIV, whilst on the WHO list, are largely endemic diseases which occur in large numbers at a constant or slowly evolving rate in many populations. TB and HIV also have a long asymptomatic phase and in most cases onset of clinical disease years after initial infection. They do not, therefore, have a large, sudden population impact with clinical cases increasing rapidly over a short period of time. This could change if these microbes are artificially engineered for rapid spread or altered latent periods.Severe acute respiratory syndrome (SARS) was an epidemic infection, in contrast to Middle East Respiratory Syndrome (MERS), which has displayed a more sporadic pattern with occasional outbreaks. For most emerging viruses which have spread globally , there are no commercially available anti-virals, and thus AMR is not relevant. However, drugs are in development for some of these, such as Ebola, which has epidemic potential.The discussion above focuses entirely on natural events. However, there is an increasing risk of unnatural pandemics due to quantum advances in science, availability of methods for engineering pathogens, open access science and global geopolitical instability. Unnatural pandemics may occur as a result of deliberate release or a laboratory accident. Our systems, governance and legislation have not kept pace with changes in science, leaving the world vulnerable. Developments in gene editing, particularly Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein-9 nuclease (Cas9), makes it possible today to engineer plants, animals, human beings and microbes. Editing of healthy human embryos, for example, is underway in Sweden, and modification of avian influenza viruses, SARS and other pathogens has already occurred. Synthetic viruses and genetic engineering of pathogens is an example of dual-use research of concern (DURC) - research intended for benefit but which may also harm humans. The capacity for biological weapons of mass destruction is vastly greater today than even a decade ago, and an unnatural pandemic may not be correctly detected as unnatural because of our inability to differentiate unnatural from natural epidemics . This caIn summary, in its present form, AMR is a gradually increasing problem with a chronic and endemic pattern. The global concern about AMR can be broken down into two separate concerns. The first is the gradually rising incidence of AMR in many bacteria including TB and many hospital or community acquired infections. This is an endemic problem, driven by patterns of antibiotic use in animals and humans, with a gradual rise in cases over time. The impact on a population level can be compared to other endemic diseases such as diabetes or heart disease. The second concern is AMR as a compounding factor in a pandemic infection. Whilst the term pandemic is widely misused, technically it refers to a global epidemic in which there is a rapid rise in cases over time, and therefore an immediate, sharp impact on society and its systems. If AMR affected a pandemic, it would contribute to overall death and disability on a sudden and large scale. Of all currently known infections with pandemic potential, influenza is the most probable cause of a major human pandemic. There are other potential causes, but many of these have no available drugs for treatment. In addition, the technology exists today to change living organisms from their natural state into an unnatural state, which may result in previously unknown types of pandemics. The exact impact of AMR in a pandemic needs to be quantified for specific diseases, and we have provided a general formula which can be used to do so. This formula can be expanded with many more factors, but the most important determinants of impact are included.


Information about the geological timeframe during which animals radiated into their major subclades is crucial to understanding early animal ecology and evolution. Unfortunately, the pre-Cambrian fossil record is sparse and its interpretation controversial. Relaxed molecular-clock methods provide an alternative means of estimating the timing of cladogenesis deep in the metazoan tree of life. So far, thorough molecular clock studies focusing specifically on Metazoa as a whole have been based on relatively small datasets or incomplete representation of the main non-bilaterian lineages (such as sponges and ctenophores), which are fundamental for understanding early metazoan evolution. Here, we use a previously published phylogenomic dataset that includes a fair sampling of all relevant groups to estimate the timing of early animal evolution with Bayesian relaxed-clock methods. According to our results, all non-bilaterian phyla, as well as total-group Bilateria, evolved in an ancient radiation during a geologically relatively short time span, before the onset of long-term global glaciations . Importantly, this result appears robust to alterations of a number of important analytical variables, such as models of among-lineage rate variation and sets of fossil calibrations used. Most animal phyla appear in the fossil record in a relatively short period during the Cambrian (541–485 million years ago [Ma]), the so-called “Cambrian explosion”4. However, these Cambrian fossils already exhibit complex morphologies, supporting the idea that animals must have evolved some time during the Proterozoic (2500–541 Ma). Indeed, it is now widely agreed that animals existed at least during the later stages of the preceding Ediacaran period (635–541 Ma), as evidenced by trace fossils and some body fossils with likely metazoan affinities7. Unfortunately, the older fossil record is relatively scarce with respect to possible animals, and most findings are being controversially discussed8, which prevents reliable inferences about the origination times of the five main lineages. Therefore, molecular palaeobiological approaches10 might aid in deciphering early animal evolution by means of divergence time estimation from genomic data of extant species (molecular clock studies).Our understanding of the origin and evolution of animals (Metazoa) and their major subgroups would be greatly enlightened by better knowledge about the timing of diversification early in their history. Metazoa comprises five main lineages . Furthermore, the dataset they used for dating their trees is comparatively small, being composed of sequence data from seven proteins only (~2000 amino acid positions). Thus, the results of Erwin et al.12 remain to be tested with a more complete taxon sampling and phylogenomic-scale data. Philippe et al.18 used a 128-protein phylogenomic dataset  to reconstruct animal phylogeny. This dataset includes all non-bilaterian phyla and classes, a selected sample of representatives from all three bilaterian supergroups , as well as a suite of non-metazoan opisthokonts as outgroups. Supporting a well-resolved phylogeny, this dataset appears well suited for investigating the timing of deep metazoan phylogeny. However, in-depth molecular clock studies utilizing this dataset have thus far been lacking. Here, we present molecular clock analyses of the Philippe et al.18 dataset under a variety of analytical conditions. Our results confirm previous molecular-clock estimates of an early-mid Neoproterozoic  origin of crown-group Metazoa, before the onset of long-lasting global glaciations, the Sturtian and Marinoan “Snowball Earths” of the Cryogenian (720–635 Ma21). In contrast to previous studies, however, our results suggest that not only crown-group Metazoa, but all non-bilaterian phyla , as well as total-group Bilateria, originated before the Sturtian, probably within a geologically relatively short time span. Importantly, this main conclusion is robust to a number of major assumptions that can drastically influence the outcome of molecular clock studies.In a highly cited molecular clock study, Erwin 22. In order to assess the robustness of our age estimates to a number of prior assumptions, we ran analyses under (1) an autocorrelated and an uncorrelated relaxed molecular-clock model – the former assuming that the rate of molecular evolution of a lineage is correlated with the rate of its mother lineage, the latter allowing completely independent rates from lineage to lineage; (2) three different sets of fossil calibrations for internal nodes – one aiming at a maximum breadth of calibrations (Set A), another one excluding some potentially controversial fossils (Set B), and a third one adopted from Erwin et al.12 (Set C); (3) different prior assumptions about the age of the root of the phylogeny (=origin of crown-group Opisthokonta) – 800, 1000, and 1360 Ma; and 4) three different alternative assumptions about the phylogenetic placement of Ctenophora , which is currently a matter of debate26. More detailed descriptions of these, as well as some additional, analyses are given in the Supplementary Material.We conducted a range of relaxed molecular-clock analyses using the Bayesian Markov Chain Monte Carlo (MCMC) implementation PhyloBayesMean age estimates for the nodes of greatest interest were generally older, sometimes considerably so, under the uncorrelated model Fig. . The onlC yielding the oldest, A the youngest, and B intermediate estimates , and Ctenophora. Mean estimates for Calcarea were mid-Cambrian under all three calibration sets, with CrIs narrowest for A and widest for C. The mean estimate for Hexactinellida  was late Silurian under A, whereas under B and C, which did not constrain this node, much younger and certainly incorrect27 estimates  were obtained. The situation for crown Ctenophora was very similar: Lower Devonian with the minimum constraint enforced (A and B), and Lower Cretaceous without the constraint (C). Overall, set A gave the most plausible and precise results, especially regarding the deepest nodes, which were excessively old under sets B and C. Furthermore, the calibration set of Erwin et al.12, which we used for set C has been heavily criticized28, so results obtained under set C have to be interpreted with caution. Therefore, we focus on results obtained with calibration set A in the remainder of the paper.For the deepest nodes (Opisthokonta to Eumetazoa), all three calibration sets resulted in pre-Sturtian age estimates, with tes Fig. . The samet al.12 .Not surprisingly, for almost all nodes of major interest, age estimates were younger than those obtained under the 1000 Ma root age prior when 800 Ma was assumed for the root, and older when 1360 Ma was assumed . However, in contrast to these earlier studies, which found a more protracted diversification of non-bilaterian animals, we have inferred a striking pattern of a relatively fast radiation of these lineages and their basic subgroups, prior to the Neoproterozoic Snowball Earth periods (Fig. Using Bayesian relaxed molecular-clock dating on a phylogenomic dataset with representative taxon sampling of all five major animal lineages, we have inferred that crown-group animals have a deep pre-Ediacaran origin, which is consistent with several previous studies (reviewed in Sharpe ods Fig. , this ge37. However, claims of pre-Ediacaran animal remains are still controversial and more work needs to be done to reconcile the fossil and molecular records7. Clearly, a more thorough exploration of the Proterozoic fossil record will be necessary to obtain unambiguous evidence for pre-Ediacaran metazoans and a better picture of the morphology and ecology of the early members of the major extant animal lineages. Our results, if accurate, raise important questions such as what triggered this early radiation of animals and how did they survive Snowball Earth? However, future analyses of independent phylogenomic datasets with equally or better suitable taxon sampling, and employing yet other analytical set-ups will be necessary to further test the robustness of these results. Although reconstructing the time-line of animal evolution with high precision might not be possible with current molecular-clock methodology14, general patterns such as the one reported here can certainly be detected and will provide a much-needed framework for future research on the origin and early evolution of the Metazoa.Our estimates are consistent with some palaeontological and geochemical findings interpreted as evidence for Tonian to Cryogenian animal lifeSUPPLEMENTARY INFORMATION