The prognosis of mitral valve replacement is an important clinical issue and may produce unexpected mortality rates if not properly addressed. The postoperative examination results have important prognostic implications. This study was designed to determine the prognostic value of phosphocreatine and inflammatory markers after mitral valve replacement.  Comparison and analysis of the data obtained using SPSS software. The computer retrieved PubMed, Science Citation Index (SCI), Embase, VIP, CNKI, CBM, and Wanfang database and manually retrieved randomized controlled trials (RCTs) published at home and abroad on the central muscle protection role of creatine phosphate in heart valve replacement, and the search period was established until February 2018. Two random literature reviewers independently screened the literature and extracted data, using Review Manager (RevMan) (Computer program), version 5.3 . RevMan software version 5.0 assesses the risk of bias for inclusion in studies. The software performs a meta-analysis of the obtained data. P < 0.00001), (2) the creatine kinase isoenzyme level in the venous blood of the phosphocreatine group after 24 h of aortic blocking was significantly lower than that in the control group , and (3) Troponin I levels were significantly lower in the intravenous creatine group than in the control group 24 h after opening of the aortic block .  Ten RCTs with a total of 464 participants were enrolled. The meta-analysis results showed that (1) elevated creatine kinase levels often predict a better prognosis after mitral valve replacement (RR = 1.36, 95% CI: 1.22 to 1.52,  Creatine phosphate and inflammatory factor have good predictive value for the prognosis of mitral valve replacement. Because of its insidious onset and long course of disease, it is difficult to detect in time, which affects early treatment and often progresses to chronic or even severe valvular heart disease [Mitral stenosis usually progresses slowly, with an average annual decrease in mitral orifice area of 0.01 cm disease . It can  disease , 5.Reconstruct valve function to avoid accidental death.These are the following indications: (1) mitral stenosis and severe calcification; (2) mitral stenosis, severe valve contracture, and severe subvalvular lesions that cannot be repaired by surgery, (3) mitral stenosis and insufficiency, which cannot be solved by surgery; and (4) simple mitral insufficiency that cannot be corrected by surgery .These are the following contraindications: (1) Rheumatic activity was not controlled or controlled for less than 3 months; (2) patients with heart failure complicated with myocardial ischemic damage, such as advanced patients with aortic stenosis; (3) patients whose liver, kidney function, or general condition is too poor to undergo surgery; and (4) patients with bacterial endocarditis with sepsis and multiple infections should not be operated on .Selection of artificial heart valve is as follows: what kind of artificial valve should be used in valve replacement surgery that should be analyzed according to the specific situation? We should consider the patient's age, occupation, physical strength and mental state, the patient's opinions on valve selection, the patient's myocardial condition, and whether the patient can receive long-term anticoagulant therapy , 9.Patients with mechanical valve replacement need to take anticoagulants for life , regularly go to the hospital to recheck Pt, and monitor the anticoagulant effect. The patients with biological valve replacement were gradually reduced after taking anticoagulants for 3 ~ 6 months and stopped within 1 ~ 2 weeksSpecial problems during anticoagulation: in case of black stool, hematuria, hemoptysis, gum bleeding, syncope, hemiplegia or sudden dull pain in precordial area, go to the nearest hospital for examination and treatment immediately. Injuries should be avoided. In case of injury or surgery, the receiving doctor should be informed that anticoagulants are being taken. If pregnancy is needed, contact the hospital in advance to adjust the anticoagulation schemeAntibiotics should be used in time during tooth extraction, urethral dilatation, catheterization, and enteroscopy because bacteria are easy to reproduce at the artificial valve, resulting in artificial valve endocarditis. Infectious diseases such as skin furuncle and tonsillitis should be treated in timeAfter heart valve surgery, the recovery of cardiopulmonary function generally takes 6 months to 1 year. Therefore, it is generally considered to resume work after 6 ~ 8 months of rest, and the labor intensity and workload increase gradually. Patients with cardiac function reaching grade I can gradually stop cardiac stimulants and diuretics and return to normal work, but strong physical labor should be avoided. Patients whose cardiac function cannot reach grade I should continue to take cardiac stimulants and diuretics and engage in less intensive work according to their own conditionsRandomized controlled trial (RCT) is as follows: adult patients undergoing heart valve replacement for the first time in the context of external circulation, ethnicity, nationality, gender, and course of illness are not limited. Outcome index is as follows: (1) the self-synchronization rate of visceral electrical activity in the operation center was better, (2) aortic blockade opens 24 h after the phosphate creatine group of venous blood creatine kinase isoenzyme  level, and (3) aortic blockade opens 24 h after the creatine phosphate group of venous blood myocardial troponin I  level –12.These are the following factors of the exclusion criteria: (1) interventions do not meet, (2) the test object does not match, (3) studies that are repeatedly published or have similar information, (4) overview and summary, and (5) documents for which data cannot be extracted.English database retrieval strategy is as follows: subject words and free words are used for retrieval. The search words are as follows: “rheumatic heart disease,” “creatine phosphate,” “mitral valve pair,” “valvuloplasty,” and “valve replacement”. Chinese key words are as follows: “rheumatic heart disease,” “mitral valvuloplasty,” “creatine phosphate,” “mitral valve replacement”, and the references included in the literature and the compilation of academic conference papers, etc.Z-test was used to infer the combined effect. The statistical inference of heterogeneity between included research results was Q-test and I2. Funnel plot was used to detect publication bias, and Begg rank correlation and egger linear regression were used to quantitatively detect publication bias. The difference was statistically significant with P < 0.05.The literature quality was evaluated according to the bias risk table recommended by Cochrane Handbook 5. The included studies were retrospective clinical studies and nonrandomized blind method; so, there is the possibility of selective bias. RevMan 5.3 software and Stata 12.0 software provided by the Cochrane Collaboration Network are used for statistical analysis. Odds ratio (or) or relative risk (RR) and its 95% confidence interval (95% CI) are used as effect analysis statistics for classified data, and mean difference (MD) or standardized mean difference (SMD) and its 95% CI are used as effect analysis statistics for quantitative data. μL and 0 34 mol/L (8-hydroxyquinoline 50 μL and 0 32 mol/L of dimercaptopropanol 25 μL). No anticoagulation is required for the detection of CRP and IL-6. Centrifuge at 3000 rpm (the high-speed centrifuge adopts Beckman company of Germany) for 5 min. The separated serum samples are stored at -30°C for batch detection. Creatine phosphate and IL-6 are analyzed by radioimmunoassay ; CRP was measured by rate scattering turbidimetry (kit provided by German Deling company). Olympus AU5400 automatic biochemical analyzer is operated by professional inspection technicians according to reagent operation instructions. As noted above, these inflammatory blockages may be important for studying the recovery of the heart after changing behaviors. Infection is often essential for increasing the chances and improving over time for recovery and early detection of movement. With these unique characteristics and inflammatory features, it is easy to predict the complexities mentioned in our initiatives.Preoperative routine examination is as follows: all patients underwent preoperative routine examination of cardiac color Doppler ultrasound , ECG, and chest film. Sample collection is as follows: all subjects fasted for 12 hours and fasting venous blood in lying position in the morning. For the detection of creatine phosphate, it is necessary to quickly inject it into the 4°C precooled enzyme inhibitor anticoagulant tube (containing 0.3 mol/l disodium EDTA) In literature search results after resisting 367 articles obtained from the initial screening, 10 RCT words were finally included. A total of 464 patients were included, including 233 cases in the creatine phosphate group and 231 cases in the control group. The literature screening process and results are shown in Tables P < 0.00001), see P < 0.05), The score of postoperative quality of life in this group was significantly higher than that before operation (P < 0.05). With the increase of age, the function of various organs of the human body appears progressively degraded. Therefore, sensitivity analysis of the studies with the largest mean age excluded from the analysis showed that the difference in pooled effect values of the largest average age of patients was still statistically significant ((RR) = 1.40, 95% CI: 1.25-1.58, P < 0.00001). Literature of 9 RCTs was included . There wI2 = 93%), using a random effects model, and the results showed that venous blood levels in the creatine phosphate group after opening 24 h after the opening of the aortic block were lower than in the control group , see Aortic blockade after 24 h of venous blood CK-MB level was included in 3 RCTs. The results between the groups were statistically heterogeneous  had no significant change within 2 weeks after operation and increased significantly by 6.21% after half a year, indicating that the left ventricular systolic function of these patients recovered well after operation. LVEF decreased by 3.05% within 2 weeks and increased by 7.69% half a year after operation, but the difference was not statistically significant . The reaIn the surgical treatment of rheumatic heart disease, mitral valvuloplasty has better short-term and long-term outcomes than replacement . These aAt present, the research of transcatheter mitral valve replacement still stays in the research of instruments and early clinical effects, and there is no powerful research that can really promote transcatheter mitral valve replacement to practical clinical application . AlthougCreatine phosphate is an important effector molecule in response to the activation of energy metabolism system . This stThe role of inflammation theory in the pathogenesis of atrial fibrillation is a research hotspot in recent years. CRP represents an important indicator of inflammatory response and has high sensitivity. It can indicate the degree of local or systemic tissue inflammation, which has been used in clinical practice. IL-6 is a multifunctional cytokine that regulates immune response and inflammatory response . This stCorrelation analysis showed that creatine phosphate, CRP, and IL-6 were positively correlated with left atrial diameter and negatively correlated with LVEF, further suggesting that creatine phosphate and inflammatory cytokines play an important role in the process of cardiac insufficiency. It is found that creatine phosphate can promote inflammatory response and increase the expression of proinflammatory cytokines. There is a synergistic effect between phosphocreatine and inflammatory response: phosphocreatine promotes inflammation, and inflammation itself can also activate phosphocreatine, thereby increasing the expression of Ang II. However, it should be noted that in order to explore the exact role of creatine phosphate and inflammatory response in the pathogenesis of valvular atrial fibrillation, further large-scale basic and clinical research is needed.


Results suggest that electrical conductivity is a driving force of system heterogeneity. Such spatial heterogeneity within the Puquios is likely to be expanded with temporal observations incorporating expected seasonal changes in electrical conductivity. The complexity of these Andean ecosystems may be key to their ability to persist in extreme environments at the edge of habitability.Reputed to be the driest desert in the world, the Atacama Desert in the Central Andes of Northern Chile is an extreme environment with high UV radiation, wide temperature variation, and minimum precipitation. Scarce lagoons associated with salt flats  in this desert are the surface expression of shallow groundwater; these ponds serve as refugia for life and often host microbial communities associated with evaporitic mineral deposition. Results based on multidisciplinary field campaigns and associated laboratory examination of samples collected from the Puquios of the Salar de Llamara in the Atacama Desert during austral summer provide unprecedented detail regarding the spatial heterogeneity of physical, chemical, and biological characteristics of these salar environments. Four main lagoons (‘Puquios’) and more than 400 smaller ponds occur within an area less than 5 km Study of modern extreme environments suggests that variability in physico-chemical conditions can induce differences in microbial morphology, ecosystem function, and mineral deposition9. Detailed characterization of the response of ecosystem function and microbe-mineral interactions to variations in the physico-chemical conditions that are commonly observed in extreme environments are necessary for accurate interpretations of depositional setting and potential biosignatures in the rock record11.Life on Earth can thrive in almost every ecological niche, and many extremophiles prosper in the harshest of environments33. An example of a gypsum-depositing system in an extreme Andean environment is a series of lagoons locally known as Puquios in the Salar de Llamara, located in the Atacama Desert. The Salar de Llamara is located in one of the main endorheic basins in the Pampa del Tamarugal, Central Depression, of the Norte Grande de Chile36. The Puquios are variably sized depressions filled with ground water. The Pampa del Tamarugal is the largest and last base level before the Pacific Ocean for the sedimentary fill and water input provided by the High and Preandean Cordilleras. The sedimentary fill consists of detrital sediments and volcanic rocks that can reach up to 1300 m thickness37. The Puquios, in this arid to hyperarid environment of the Atacama Desert, are subject to a complex interplay of physical, chemical, geological, and biological processes. Known environmental pressures in the Salar de Llamara include high UV irradiation, extreme aridity, and significant fluctuations of temperature and salinity39, all of which contribute to the characterization of the Puquios as a dynamic extreme environment40. Despite these harsh environmental conditions, the Puquios support the development of a variety of biotic communities and exhibit diverse styles of gypsum deposition. Previous studies of these ecosystems have typically focused either on the brine chemistry, or the microbial communities, or the deposition of carbonate and evaporitic minerals43, with limited holistic characterization across disciplines. In order to more fully understand these extreme settings, a multidisciplinary approach that captures physical, chemical, geological, and biological observations is needed.Extreme environments hosting microbial life are common in the Andean regions of South America. Studies of more than 40 microbial ecosystems forming in harsh conditions in Chile, Argentina, and Bolivia document a wide range of geological, chemical, biological and mineralogical processesThis paper presents data from multidisciplinary studies of the Puquios in the Salar de Llamara during the austral summer. Results provide unprecedented detail regarding the spatial heterogeneity of physical, chemical, and biological characteristics of these salar environments. Our observations include more than 1285 in situ measurements of electrical conductivity in the surface and bottom waters and descriptions of free-living biota in each lagoon, endolithic microbial communities, and mineralogic and morphologic analyses of bottom types. Spatial distributions of biological and geological features observed throughout the system appear to be linked to gradients in electrical conductivity. The high degree of spatial heterogeneity in all measured parameters highlights the need for multi-disciplinary studies across space and time to understand ecosystem function and mineral deposition in extreme environments.13. This salar is located in the distal part of the Arcas Alluvial Fan that provides most of the basin input44. The hyperaridity of the Central Depression has fostered the deposition of evaporitic sediments since at least the Middle Miocene36, and is controlled by the geologic and oceanographic setting47. Rainfall is extremely low, estimated to be less than 1 mm in the Atacama Desert core48 as a result of the rain-shadows cast by both the Coastal Cordillera and the High Cordillera. Furthermore, evaporation rates significantly exceed rainfall, with up to 90% of rainfall in the Western Cordillera lost via evaporation and runoff49. The permanent surfacing of sub-terrain waters is recharged by a combination of summer monsoons (Invierno Altiplánico or Invierno Boliviano) in the High Cordillera52 that continuously drain through the alluvial fan systems punctuating the eastern margin of the basin54 with occurrences of coastal dripping fog56. The combination of these hydrological processes associated with the alluvial fan systems and the geomorphological characteristics at the boundary between the Coastal Cordillera and the Pampa del Tamarugal allows the shallow ground water levels to create the small lagoons called puquios with their biological communities. Brines in the Puquios are managed by SQM Ltd.; environmental protection measures were enacted in 2012 to preserve brine chemistry and water levels in the salar environment58.The Puquios Fig.  are loca2 and a maximum depth of 50 cm. A multitude of small ponds exist between the main lagoons of Puquio 1 and 2 that are collectively referred to as “the Transition Zone”. The Transition Zone contains nearly 400 small lagoons of various water turbidities, sizes, and depths (maximum depth recorded 80 cm). Puquio 2 is largest lagoon in the Salar de Llamara with very low-turbidity brines, an area of 4650 m2 and a depth less than 1 m. The main lagoon of the Puquio 3 system has an area of 1660 m2, elongated north–south, surrounded by a number of smaller, peripheral ponds. Water in Puquio 3 is clear, whereas the shallow peripheral ponds often have milky-white to peach-colored water. Puquio 4 consists of one main lagoon (1485 m2) elongated north–south, surrounded by numerous peripheral ponds, mostly to the east. The main lagoon of Puquio 4 appears turquoise in color, and the water is clear, whereas the surrounding ponds also often have milky-white to peach-colored water similar to those adjacent to Puquio 3.The Puquios system is characterized by four main lagoons Figs. b, 2, pluResults from high resolution studies of electrical conductivity of brine waters, free living biota, microbial endoliths, and bottom types are presented below.More than 1285 in situ measurements of electrical conductivity EC, Fig.  were colWithin the system as a whole, EC exhibited a high degree of spatial heterogeneity between Puquios Fig. . The lowSpatial heterogeneity of surface EC values was also commonly observed within individual lagoons. The main lagoons of Puquios 2, 3, and 4 contain ranges of EC values greater than 89.5, 89.6, and 138.0 mS/cm in their surface brines  was observed in Puquio 2 , phytoplankton , zoobenthos (invertebrates found on the bottom or buried within the first few centimeters of substrate) and zooplankton (invertebrates in the water column). The first two groups, which are photosynthetic primary producers, are represented mainly by diatoms (50 species) and by the less diverse cyanobacteria (8 species). The primary and secondary consumers present on the bottom and in the water column are represented by 8 taxa: arachnids (spiders), annelids (worms), branchiopods (brine shrimps), coleopterans (beetles), copepods, dipterans (flies), hexapods (springtails), gastropods (snails) and odonates (dragonflies). Several of these taxa have complex life cycles and thus occur in different ontogenetic stages . Some, like dipterans and odonates, only spend their larval phase in the water while some, like the brine shrimp Denticula, Staurosira and Nitzschia are the most represented genera of diatoms on the bottom of Puquios 1, 2 and 4, respectively, while the water column is dominated by Cyanophyta in Puquio 1 and Staurosira and Nitzschia in Puquios 2 and 4, respectively. Although, the taxa recorded in the four Puquios are commonly observed in other high Andean saline systems60, the particular dominance of certain taxa in different puquios is intriguing. For example, the genus Nitzschia contains many pollution-tolerant species61 that have been used as indicators of water quality62.All four assemblages of biota exhibited ample variability in several ecological descriptors , was conducted  and gamma  were predominant within the Proteobacteria phylum.The bacterial communities were assessed in subaerial gypsum structures and the adjacent brines Fig. a. The en−, and SO42−, Pearson correlation > 0.9; Supplemental Table + concentrations (Pearson correlation > 0.8)  analysis grouped the brine bacterial communities related to brine chemistry . Desulfobacterales was observed among the sulfate reducers which has been reported previously in the subaqueous structures14. Desulfobacterales was registered as a dominant, active sulfate reducing bacteria and primary hydrogenotrophs in the upper layer of a microbial mat63.The metabolisms from the predicted metagenomes by PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) suggests that there are key metabolic differences between E1 layers and the free-living communities in the brines, and also between the E1 layers from each Puquio. The occurrence of the sulfur oxidation pathway appears to be dominant in both brines and subaerial structures Fig. e. PhototA range of different bottom types Fig. , S6 and Bottom types in the Transition Zone were highly variable, grading from flocculent sediment to a variety of orange, brown, and black microbial mats, with various morphologies Fig. , S6a–c. Euhedral gypsum spar characterizes the bottom type of the main lagoon of Puquio 2 as well as the Transition Zone lagoons to the southwest Figs. a,b, S7d.The main lagoon of Puquio 3 was dominated by pinnacles Fig. c,d, S7e The dominant bottom type in Puquio 4 was gypsum spar Figs. y, S6e,f.Heterogeneity within and between Puquios is reflected across all parameters investigated, including electrical conductivity, diversity and abundances of free-living biota, community structure and metabolisms of endolithic microbial ecosystems, as well as the dominant lagoon bottom types and associated minerals. For the most part, variations in these ecological, microbiological, and mineralogical datasets appear to follow gradients of electrical conductivity, which exhibits heterogeneity on both the scale of the salar environment as a whole, as well as within single lagoons. As summarized in Fig. 43. EC measurements in these previous studies, as well as in situ measurements of total dissolved solids (TDS) conducted by Otálora et al.22 documented a trend of increasing EC moving from SW to NE in the P1-TZ-P2 region, which is consistent with hydrogeological models of flow direction in the area58. Variations in the absolute values of EC measurements in the P1-TZ-P2 region between the data presented here and those reported previously are likely a function of the hydrogeology of the system, where seasonal variations in primarily evaporation, as well groundwater influx and local injection influence water levels and EC values66. Importantly, the persistence of a consistent spatial gradient over more than a decade since the initial measurements collected by Otálora et al.22 implies the hydrogeological factors driving gradients in the chemistry of the system are likely resilient to perturbation.Gradients in electrical conductivity are the predominant unifying factor that drives the ubiquitous spatial variability throughout the lagoons Fig. . New ins43 during summer months include an EC measurement (191 mS/cm) that was higher than our more recent observations in Puquio 4 . Other abundant diatoms in these puquios include Fragilariales , which are common in aquatic environments of the arid Andes of Chile and considered indicators of early successional stages74. On the other hand, cyanobacteria in the puquios occur in highest abundance in the shallowest lagoons with lower EC values (Puquio 1 and Puquio 3), where they conduct anoxygenic photosynthesis75 and share the ability to use elemental sulfur for anaerobic dark respiration with many archaeobacteria76, thus, favorably exploiting the local conditions in these puquios. Diversity indices for both phytoplankton and phytobenthos, show negative linear relationships with EC, as less halotolerant species are excluded with increasing EC.Gradients in electrical conductivity appear to drive spatial distributions of abundances and diversity of the free-living biota in the Puquios ecosystem Fig. . PrimaryArtemia franciscana in all four main lagoons. Originally from North America, the presence of this species in the Puquios, and northern Chile in general, is strongly linked to bird migratory flyways77. The cyst of Artemia is highly resistant to extreme environmental conditions, while the motile stages are among the best osmoregulators known78, demonstrating their adaptability to the harsh environmental conditions that characterize extreme environments. In contrast, zoobenthos abundance showed no linear relationship with EC, with the higher abundances in Puquios 1 and 4 and lower abundance in 2 and 3  were more variable than primary producers in their response to increasing electrical conductivity Fig. . Zooplan81 The E1 layer of low EC puquios was associated with higher relative abundances of nitrate and sulfate reducers and sulfur oxidizers, whereas the E1 layer of high EC puquios was associated with higher abundances of halophilic and halotolerant oxygenic and anoxygenic phototrophic genera  shows a general decreasing trend with EC  and differences in the brine bacterial communities were correlated to electrical conductivity in the principal coordinate canonical analysis . However, our high-resolution assessment indicates that within these major classifications, a variety of both microbial and crystalline bottom types contain a diverse suite of minerals and morphologies that occurs within the gradient of EC values. This observation suggests the system is more heterogeneous than previously appreciated. Similar analyses conducted in other extreme environments have demonstrated the utility of such high-resolution observations, where spatial context of the diverse bottom types with environmental parameters provides insight into factors that impact and often segment these systems68. Within the Puquios, the gradient in EC values appears to be the overarching mechanism that subdivides the bottom type morphologies and minerals precipitated. Where EC values are relatively low, as in Puquio 1 . As a result, these findings suggest that variability in the morphological expression of bottom types is a useful visual representation of both the environmental gradients and likely spatial heterogeneity in mineral deposition within these ecosystems.In contrast, when brines have relatively high EC values, such as those observed in Puquio 2 and in Transition Zone ponds adjacent to the southwestern margin of Puquio 2 Figs.  the pond41. Considering that EC has an indirect correlation with surface water level in the Puquios (R2 = 0.50–0.83), seasonally variable water levels in Puquio 1 may drive the relationship between EC and microbial community structure in Puquio 1, distinguishing it from similar endoevaporitic communities near Puquios 2, 3 and 4. Similar seasonal differences in anaerobic Deltaproteobacteria abundances were observed between summer and winter samples in a previous study41 and between the depths analyzed in subaqueous niches in March 201214.The dynamic interplay between chemical, biological and geological aspects of the system may foster adaptability in community structure and function. For example, the Invierno Altiplánico that occurred in the Atacama Desert in January–February 2017 is hypothesized to have impacted both water levels and microbial communities in Puquio 1. Higher water levels after the Invierno Altiplánico likely filled the pore spaces of the subaerially exposed structures, reduced available oxygen in the environment that fosters endoevaporitic microbial communities, and produced the observed enrichment in the anaerobic microbial population in Puquio 1 samples. The surface water level therefore is an important determinant of the shift from a subaerial to a subaqueous lifestyle as was previously observed93, we hypothesize that heterogeneity similar to that observed in the Puquios is likely to be a common characteristic of extreme environments and that seasonal environmental variability, changes in electrical conductivity driven by seasonal cycles of evaporation, and groundwater flow are likely to impact all of the ecological niches to varying degrees at different times of the year. This concept is critical when characterizing such ecosystems, since a single sample is likely not representative of the entire system. As a result, more attention should be given to data collection through space and time when interpreting the significance of biogeochemical processes in these extreme environments.Given the reported diversity of water chemistry, biota, minerals, and structures, of other microbial Andean ecosystems within Chile, Bolivia, and Argentina94. These observations suggest that metabolic redundancy and diversity inherent in these biological communities may contribute to the resilience of the system to harsh poly-extreme conditions. Moreover, seasonal changes in precipitation, evaporation, temperature, and climatic conditions drive temporal variability in environmental conditions58 that are at least as extreme as the spatial heterogeneity documented in the Puquios. Such spatio-temporal heterogeneity presents significant challenges to survival in the Puquios. Thus, we propose that heterogeneity in all aspects of the Puquios system may lead to ecological flexibility, including functional redundancy and adaptability95, both of which may be fundamental for the ecosystem to thrive in some of the harshest conditions on Earth.In summary, the diagram shown in Fig. Our study documents a previously unrecognized degree of spatial heterogeneity in water chemistry, free living biota, microbial communities and bottom types in the Puquios system. Biological and geological variability appear to be predominantly driven by heterogeneity in electrical conductivity. Results demonstrate the importance of high-resolution measurements, collected across multiple disciplines, from extreme environments that are inherently heterogeneous. Moreover, it is likely that observations from a single season do not capture the full range of variability, and enhanced spatio-temporal heterogeneity is expected as seasonal changes in environmental conditions fluctuate. Inherent multidisciplinary heterogeneity may be key to the success of these microbial ecosystems, providing flexibility that allows them to reorganize after disturbances and persist in extreme conditions at the edge of habitability.2 cylindrical plexiglass tube was gently introduced into the top bottom 1 cm layer of the substrate and all material collected stored into a 15 mL Falcon tube and fixed using 2% Lugol. Zoobenthos samples were collected using a 1256 mm2 cylindrical plexiglass tube that was also introduced into the top 1–2 cm layer of substrate and all collected material stored into an 80 mL plastic container and fixed with 5% diluted formalin. Phytoplankton and phytobenthos quantification analyses considered APHA95 methods 10200 C, D, E, F and 10300 C, and for identification several sources were consulted (e.g.105). Zooplankton and zoobenthos quantification analyses considered APHA95 methods 10200 G and 10500 A, C and Woelf et al.106 and for identification several specialized sources were consulted (e.g.112). A linear regression analysis of the relationship between abundance as well as diversity (Shannon index H′) and electrical conductivity was completed using Prism version 8.4.2. from GraphPad Software .Biotic survey of water column and benthic organisms was performed at 5 sites in austral summer 2017 within each of the 4 Puquios. At each site 3 replicate samples were obtained. For zooplankton and phytoplankton 36 L of water was passed through a 35 µm and 60 µm sieve respectively by means of an electrical immersion pump powered by a portable 12 V battery. Each sample was summarized and introduced into a 50 mL Falcon tube and fixed using 2% Lugol for phytoplankton and 5% diluted formalin for zooplankton. For phytobenthos, an 875 mm113. The filter was then folded and placed in a tube with a hypertonic lysis buffer solution  and was kept at − 20 °C up to later processing. DNA was obtained using the AllPrep Qiagen RNA/DNA Isolation Kit . The extraction yield and DNA purity were determined by means of gel electrophoresis and UV–Visible spectrophotometry .Bacterial communities were collected from subaerial gypsum structures at 3 sites from each of the four Puquios (n = 12) using a 20 cm sediment core device in March 2017. A sample of the microbial mat inhabiting the gypsum structure was collected from the E1 ‘green layer’. A 1 L brine sample was collected from the water surrounding each subaerial gypsum structure for comparison between the planktonic microbial community and the microbial mat community. The E1 from each core sample of the subaerial gypsum structures was isolated in the lab for DNA extraction and the brines were filtered by a nitrocellulose membrane with a 0.22 µm pore size  to collect the cells as it was reported before114 and 534r (5′-ATTACCGCGGCTGCTGG-3′)17 for bacteria with a barcode on the forward primer. PCR was performed using the HotStarTaq Plus Master Mix Kit  under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which we performed a final elongation step at 72 °C for 5 min. PCR products were visualized in 2% agarose gel to determine the success of amplification and the relative intensity of the bands. Multiple equivalent samples of PCR products were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beadsto prepare a DNA library by following the Illumina TruSeq DNA library preparation protocol. Sequencing was performed at MR DNA  on a MiSeq, following the manufacturer’s guidelines. The sequence data were processed using a proprietary analysis pipeline (MR DNA) with the following steps: the barcodes were eliminated from sequences, then sequences < 150 bp and those with ambiguous base calls were removed. Then, sequences were denoised, OTUs generated and chimeras removed. The reads were added to the Sequence Read Archive (SRA) National Center for Biotechnology Information (NCBI), (http://trace.ncbi.nlm.nih.gov/Traces/sra/). Project numbers—BioProject accession: PRJNA659028 ; BioSample: SAMN15924840 ; SRA: SRR12536357 .The V1-V3 variable region of the 16S rRNA gene were amplified in a PCR thermocycler using the primers 27f. (5′-AGAGTTTGATCCTGGCTCAG-3′)115: Raw sequences were filtered by base quality score, average base content per read and GC distribution in the reads. Reads that did not cluster with other sequences, i.e. singletons (abundance < 2) were removed. Chimeras were also removed using the UCHIME program116. The pre-processed sequences were finally grouped into operational taxonomic units (OTUs) using the clustering program UCLUST at a similarity threshold of 0.97116. The pre-processed reads were used to identify the OTUs using QIIME and aligned the representative sequences against the Greengenes core set reference database using PyNAST115. A representative sequence for each OTU was classified using RDP classifier and the Greengenes OTU database. The alpha-rarefaction was then calculated by means of the “core_diversity_analysis” command and standardized the number of sequences to the smaller sample size by means of Chao 1 (2599 sequences). The rarefacted data using the Primer-7 (Primer-E) software117 was used to determine the beta diversity and plot the main coordinates graphs. Non-metric multidimensional scaling (NMDS), Principal Coordinate (PCO) and Principal Coordinate Canonical analysis (CAP) were used to build a constrained ordination of the OTUs or groups of other taxonomic levels, based on the environmental data collected at the time of sampling113. Additionally, PICRUSt118 was used to predict functional composition of the metagenomes from 16S data.The sequences were processed further by means of the QIIME  pipeline119, respectively.Brine samples adjacent to the subaerial gypsum structures were collected so correlation to the bacterial community profiles of E1 layers could be made. Cation concentrations were measured using an atomic absorption spectrophotometer . Sulfate and chloride contents were measured by gravimetric and Mohr method59, where surface measurements are subtracted from bottom measurements. Normal stratification is then defined as bottom measurements being greater than surface measurements, and inverse stratifications are defined as bottom measurements being lower than surface measurements.In situ measurements of electrical conductivity (EC) were collected over the course of three days in austral summer 2017 using a HI 9829 multimeter . The EC probe was calibrated to known standards of EC values  each morning before the start of field sampling. Sample locations were collected with a Garmin GPSMap 78 s. For EC, the manufacturer specifications for accuracy is ± 1 µS/cm or ± 1% of the reading, whichever is larger. Measurement resolution is 1 µS/cm, and the range is 0–200 mS/cm. At each location, both a surface measurement and bottom brine measurement were collected. Surface measurements were collected first at about 5 cm depth in order to minimize disruption of any brine stratification, with the probe oriented parallel to the water surface. Bottom brines were measured second by slowly lowering the probe to the bottom of the lagoon and laying it horizontally. Bottom measurement collection was delayed until EC values stabilized, often 15–20 s. These field measurements, brine depths, and GPS locations were merged into a spreadsheet and their spatial distributions mapped in ArcGIS Pro. The data were evaluated by calculating the average and standard deviations, as well as calculating the interquartile range (IQR) using the exclusive quartile calculation in Microsoft Excel (Office 365). Stratification was calculated following the methods of BabelTo distinguish physio-chemical substrates in the Puquios from substrates that are products of benthic organisms, we adopted a multiscale approach. At the macroscale, broad facies patterns were mapped using aerial imagery; these bottom type maps were refined using comprehensive ground truthing in austral summer 2017. This mapping approach identified principal bottom types based on upper few centimeters of the substrate surface across the Puquios system  with 60 mm Silicon Drift Detector at Smithsonian National Museum of Natural History.Supplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.


Objectives: This study aims to assess the feasibility of using hemofiltration for ammonia clearance in low body weight infants with an inborn error of metabolism.Design: A study of two cases.Setting: Quaternary pediatric hospital  NICU and PICU.Patients: Infants <6 months of age with an ICD-9 diagnosis of 270.6 (hyperammonemia).Interventions: Continuous renal replacement therapy (CRRT).Measurements and Main Results: We measure serum ammonia levels over time and the rate of ammonia clearance over time. Continuous renal replacement therapy was more effective than scavenger therapy alone (Ammonul™) for rapid removal of ammonia in low weight infants (as low as 2.5 kg).Conclusions: Continuous renal replacement therapy is technically feasible in low weight infants with severe hyperammonemia secondary to an inborn error of metabolism. Pediatric inborn errors of metabolism that cause hyperammonemia pose specific technical challenges in critical care management, especially in the newborn period when presenting in extremis. During this newborn period, both urea cycle defects (UCDs) or organic acidemias commonly present with hyperammonemia. Additionally, to a lesser extent, some patients with maple syrup urine disease and fatty acid oxidation disorders can also have mildly elevated ammonia. Infants with these aforementioned diseases commonly present with decreased levels of consciousness and poor feeding, while up to 50% can also present with seizures . ApproprThe ammonia scavenging agent sodium acetate and sodium phenylbutyrate (Ammonul™) has been successful in improving outcomes for this patient population. Previous reports by Enns et al. show improved survival of up to 84% in children with hyperammonemia—a staggering improvement when directly compared to the 84% mortality that Nasogne et al. reported during the same time period for patients that did not receive sodium acetate and sodium phenylbutyrate , 3. DespFor neonates with severe UCDs or other in-born errors of metabolism, continuous renal replacement therapies (CRRT) including hemofiltration may be beneficial. Unfortunately, the use of hemodialysis for newborns with hyperammonemia is technically difficult due to vascular access and the extracorporeal circuit volume which is large in proportion to an infant's circulating blood volume of 75–90 ml/kg. Additionally, RRT requires high blood flow and dialysate rates (in ml/min) that have the potential to cause circulatory collapse and/or cardiac arrest. Hemodialysis alone is often associated with rebound hyperammonemia . ConsequWith careful considerations given to obtaining vascular access and maintaining adequate hemodynamic support, renal replacement therapy can be effective even for low birth weight infants. Our aim with this study is to assess the feasibility of CRRT in infants presenting with severe hyperammonemia. We hypothesize that dialysis is an effective method for ammonia clearance in this patient population and careful consideration for its deployment may impact overall outcomes.We conducted a retrospective cohort study in children admitted from 2009 to 2015 presenting with emergencies from inborn errors of metabolism at a single center, quaternary pediatric hospital. Local IRB approved the study design and waived the need to obtain informed consent (Washington University IRB# 201506089). Patients were enrolled with the following inclusion criteria: hospitalized infants <6 months of age AND a diagnosis of hyperammonemia secondary to an inborn error of metabolism, AND who received CRRT for ammonia reduction. We excluded any patients who received prior HD or who had CRRT for reasons other than ammonia clearance.The initial database screen was performed by one study investigator who queried the hospital's billing database for an ICD-9 code of 270.6 (hyperammonemia). This initial screen was further narrowed by two separate investigators who identified patients that met enrollment criteria for the study. Descriptive data is presented in each case as available in the electronic medical record.From 2009 to 2015, there were 138 inpatients with an ICD-9 coding diagnosis of hyperammonemia. Of those 138 patients, six received CRRT during their hospital course and two met inclusion criteria. All patient exclusions occurred as patients were >6 months of age. Clinical data include demographic and relevant laboratory data are summarized in 2 <90%. A point of care (POC) glucose was <1.11 mmol/L. The patient was given a bolus of D10 and D5 normal saline (NS) with a repeat blood glucose value 1 h later of 3.5 mmol/L. Blood cultures, complete blood count (CBC), and electrolyte panel were collected with a significant finding of a white blood cell (WBC) of 19.1 k/cumm. En route to the ED, the patient required bag mask positive pressure ventilation for shallow respirations and persistent hypoxemia. With signs of poor perfusion, posturing, and apnea the patient was promptly intubated. After intubation, she was hypotensive (30/20), requiring an additional 20 ml/kg NS fluid bolus. Additionally, she was given phenobarbital (20 mg/kg) for rhythmic tonic-clonic movements concerning for seizures. Given findings of hypoxemia, hypotension, new seizure disorder, hypoglycemia, and hypothermia, neonatal sepsis was considered with ampicillin and cefotaxime antibiotics and acyclovir given in the ED. The patient was promptly transported to the pediatric intensive care unit (PICU) and noted to have significant ammonia and lactate  levels. Other notable labs include aspartate transaminase (AST) of 61 units/L and alanine transaminase (ALT) of 24 units/L. Given a concern for an inborn error of metabolism, general surgery was consulted and placed an emergent dialysis catheter at the bedside. A 7 French apheresis catheter was placed in the right internal jugular vein with a venous cut down approach. Prior to catheter placement, vital signs were significant for: HR 209, RR 40 , T 36.1°C, BP 94/67, SpO2 94% on 2 epinephrine infusion (up to 0.1 mcg/kg/min) and given calcium gluconate boluses of 100 mg/kg secondary to hemodynamic instability after central line placement but prior to CRRT initiation.A 4 day old, Indian-American female that initially presented to an outside emergency department for poor feeding and grunting. Upon arrival to the ED, the patient was “sluggish,” hypothermic to 35.5°C, and noted to have hypoxemia with SpO2 vs. conventional dosing of only 2,000 ml/h/1.73 m2, as recommended for infants with hyperammonemia . Electrolytes were monitored at a minimum of every 4 h during high dose CRRT due to risk of electrolyte imbalance. The patient did not have any hypokalemia or worsening of hypothermia during her circuit run; asymptomatic hypophosphatemia did occur and was corrected with intravenous potassium phosphate infusions. After 10 h of CRRT, her ammonia level was <60 μg/dl and CRRT was discontinued. For CRRT initiation, the circuit was primed with cross-matched blood mixed with NS and 5,000 units/L heparin to hematocrit of 30%. High dose CRRT was prescribed, targeting dialysate and replacement fluid flow rates of 8,000 ml/h/1.73 mmmonemia . The CRRDue to significant disease related neurologic dysfunction over the 24 h post CRRT initiation, the family decided to redirect her goals of care to comfort measures and the patient died the next day. Her diagnostic postmortem work-up revealed a diagnosis of citrullinemia.2 of 13 mmHg, PaO2 of 151 mmHg, and base deficit of 26.5 mEq/L. Surgery was consulted for vascular dialysis catheter placement in the operating room; a 7 French apheresis catheter was placed in his right internal jugular vein using a venous cut down approach without complications. Prior to hemodialysis catheter placement, he received Ammonul loading dose of 720 mg (246 mg/kg) followed by a continuous infusion of 10.4 mg/kg/h.A 3 day old Caucasian male that presented to an outside hospital and was noted to be “listless with poor feeding and tone.” In initial assessment he was noted to be hypothermic at 32.5°C with delayed capillary refill and a POC glucose of 1.89 mmol/L. He was given 6 ml of d10 water with improvement in glucose to 3.39 mmol/L. He was fluid resuscitated for hypotension with 50 ml of NS for a systolic blood pressure of 68 mmHg. Blood, urine, and cerebrospinal fluid cultures were collected and he was started on empiric antibiotics with ampicillin and gentamicin prior to ICU transfer. Upon arrival to the ICU, his temperature was 36.7°C with pulse 136, respiratory rate 60, and blood pressure of 71/26 via non-invasive automated sphygmomanometer. His physical examination included a capillary refill <2 s, clear to auscultation lung fields, no presence of cardiac murmurs, and a liver palpated 2 cm below the right costal margin. He demonstrated minimal response to stimulation and did not exhibit a suck reflex. An umbilical arterial catheter and double lumen umbilical venous catheter were placed shortly after admission and 10% dextrose containing fluids were started at a GIR of 7 mg/kg/min. Initial laboratory screens included an ammonia of 623 μg/dl, ALT of 41 units/L, AST of 241 units/L, and an arterial blood gas with pH of 7.06, PaCO2  between the duration of a stage III or IV hyperammonemic coma and IQ at 12 months of age  had moderate-to-severe neurodevelopmental deficits compared to 28% (13/47) of patients with late-onset development of UCDs. When analyzing 108 of these patients, peak ammonia level during the first hyperammonemic attack was correlated to neurodevelopmental outcome. Specifically, when peak ammonia levels were <306 μg/dl , there were no severe neurological deficits; however, when peak ammonia levels were >596 μg/dl , patients either died or had severe neurological deficits . In contIn our case series, we describe two patients with similar presentations of severe hyperammonemia, due to different metabolic diseases, and differing outcomes. There are a number of similarities and key differences in disease presentations between both newborns. Both patients presented as small for gestational age, within the first week of life, and with severe dehydration. More specifically, Case A presented a day later than Case B, which may explain the severity of her clinical course including worsened initial acidosis and presenting ammonia level more than twice that of Case B. Despite these differences, both patients achieve rapid ammonia clearance. There are some additional differences between the presented two cases including the use of Arginine administration in Case A as compared to the decision to forego this therapy in Case B. First, although neither patient had a diagnosis prior to medical scavenger therapies, arginine therapy is not without hypothetical risk i.e., if patient A had arginase deficiency, then arginine would be contraindicated. However, given the unlikely diagnosis of arginase deficiency with a severe clinical presentation, particularly with such a high ammonia level, incongruence with typical presentation later in life, and without such profoundly high levels of ammonia, arginine was given in patient A.Additionally, both patients experience a rebound in ammonia levels just discontinuation of CRRT. We postulate that the scavenger therapies were not yet at steady state, potentially due to the clearance of this therapy because of high dose CRRT and the patients remained in a catabolic state causing an increase in ammonia. Furthermore, aggressive alimentation was not pursued during the period of CRRT; while both infants were started on appropriate initial GIR fluids, neither were escalated on dextrose containing fluids, started on lipid infusions, nor insulin infusions, all of which are methods to prevent further catabolism. The rebound phenomenon our patients experienced may be in part because of the kidney replacement strategy. Ammonia has a high volume of distribution, and therefore redistribution from extravascular space occurs. As this is a recognized and expected occurrence, some centers report using high-flux CRRT until ammonia levels return to goal range, and then continuing regular flow CRRT for another 12–24 h , 14. Thi2 . They found that the ammonia for both infants decreased to <681 μg/dl within 2 h, and <170 μg/dl within 10 h. Both infants had mild rebound in ammonia levels to 254 and 283 μg/dl (with the second infant having <170 μg/dl after 24 h of discontinuation of CRRT) , a common UCD, though they continued CRRT for 7 days while using blood flow rates of 25 ml/min, with a replacement and dialysate rate both at 150 ml/h. Additionally, this group did not utilize a scavenger therapy. Using their strategy, the patient's ammonia was reduced from a peak of >1, to 422 μg/dl at 24 h, and 222 μg/dl at 57 h. The patient had a rebound in ammonia 364 μg/dl after 5 days of dialysis and therefore continued on CRRT until 7 days . Further2) and Patient B weighing 2.92 kg (body surface area of 0.20 m2), the extracorporeal volume is 36 and 31%, respectively. Preparation and prevention for impending hemodynamic instability when initiating CRRT is necessary with the consideration and use of inotropes in addition to priming the circuit with diluted RBCs.The use of CRRT therapy in <3 kg infants has some important technical considerations. Care must be taken when initiating CRRT for small infants as the circuit volume is a significant percentage of their circulating blood volume. The extracorporeal circuit used for both patients in this series was 72 ml. If an estimated blood volume for this age range is 80 ml/kg, with Patient A weighing 2.52 kg  which further compounds the risk of hypocalcemia. Although we believe the risk of citrate toxicity was low in these patients because citrate is based upon blood flow (and not effluent rate) as well as allowing for higher post filter ionized calcium levels, the risk of hypocalcemia is high for the multitude of reasons detailed here. Calcium and electrolyte levels need to be monitored frequently, and both patients in this series needed boluses of calcium gluconate on top of the calcium infusions given for reversal of citrate anticoagulation.Our case series has some limitations. Although 138 patients diagnosed with hyperammonemia were screened for this study, only two met inclusion criteria. The most likely explanation for this is sampling bias from the retrospective nature of the study. As a result, we are only able to make definitive conclusion on the logistics of clearing ammonia in low birth weight infants via CRRT as opposed to drawing conclusions on therapeutic efficacy. For instance, despite adequate ammonia clearance, Patient A did not have meaningful neurologic recovery, which we attribute to both the severity of her enzyme deficiency and her significant disease process at the time of presentation. In comparison, the use of ammonia scavenging therapies in conjunction with the use of CRRT rapidly decreased the ammonia levels in Patient B who subsequently survived his hospitalization. This difference in patient outcome is likely secondary to differences in enzyme deficiency and severity of illness at presentation.Nonetheless, in this case-series, we show that CRRT is technically feasible in low weight infants with severe hyperammonemia secondary to inborn errors of metabolism. Undeniably barriers to ammonia clearance are logistical and primarily involve technical challenges associated with patient access and hemodynamic instability—placement of a large enough catheter to perform dialysis at high blood flow rates as well as careful monitoring of hemodynamics given the high ratio of extracorporeal volume to patient blood volume. Further studies emphasizing earlier diagnosis and rapid deployment of CRRT across centers will likely yield the greatest improvements in morbidity and mortality for this patient population. Additionally, future studies evaluating rapid CRRT initiation for severe hyperammonemia secondary to inborn errors of metabolism are needed to help clarify aspects of care teasing out timing of initiation, cases that might be ammendale to scavenger therapy alone, and prognostic factors of worsened outcomes for this patient population.CM conceptualized and designed the study, carried out the initial analyses, drafted the initial manuscript, and approved the final manuscript as submitted. CW designed the data collection instruments, coordinated and supervised data collection, critically reviewed the manuscript, and approved the final manuscript as submitted. DG, TD, and CM assisted in study design, critically reviewed the manuscript, and approved the final manuscript as submitted. KR conceptualized and designed the study, reviewed and revised the manuscript, and approved the final manuscript as submitted. All authors contributed to the article and approved the submitted version.KR is supported by the National Institutes of Health NIGMS 5K08GM129763.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Children and young adults with single ventricle (SV) heart disease frequently develop heart failure (HF) that is intractable and difficult to treat. Our understanding of the molecular and biochemical reasons underlying this is imperfect. Thus, there is an urgent need for biomarkers that predict outcome and provide a rational basis for treatment, and advance our understanding of the basis of HF.We sought to determine if a metabolomic approach would provide biochemical signatures of HF in SV children and young adults. If significant, these analytes might serve as biomarkers to predict outcome and inform on the biological mechanism(s) of HF.We applied a multi-platform metabolomics approach composed of mass spectrometry (MS) and nuclear magnetic resonance (NMR) which yielded 495 and 26 metabolite measurements respectively. The plasma samples came from a cross-sectional set of young SV subjects, ages 2–19 years with ten control (Con) subjects and 16 SV subjects. Of the SV subjects, nine were diagnosed as congestive HF (SVHF), and 7 were not in HF. Metabolomic data were correlated with clinical status to determine if there was a signature associated with HF.There were no differences in age, height, weight or sex between the 3 cohorts. However, statistical analysis of the metabolomic profiles using ANOVA revealed 44 metabolites with significant differences between cohorts including 41 profiled by MS and 3 by NMR. These metabolites included acylcarnitines, amino acids, and bile acids, which distinguished Con from all SV subjects. Furthermore, metabolite profiles could distinguish between SV and SVHF subjects.These are the first data to demonstrate a clear metabolomic signature associated with HF in children and young adults with SV. Larger studies are warranted to determine if these findings are predictive of progression to HF in time to provide intervention. The origins of HF in children and young adults are strikingly different from those in adults and most often involve structural abnormalities in heart formation , SV patients (n = 7), and SVHF (n = 9). We hypothesized that we would be able to discern a more detailed metabolic phenotype of SV patients at a younger age using a more comprehensive metabolomics approach, and that we could find unique signatures for the SVHF patients. Our goal was to better understand the potential metabolic derangements in SV and SVHF, and search for biomarkers that may be used to identify SV patients at risk for progression to HF.These results are the first to show metabolic derangements in children and young adults with SV, and more importantly, identify metabolomic profiles associated with HF in this group. This has not been done before now. Expansion of these findings will allow greater understanding of the basic mechanisms underlying HF and allow development of metabolomic profiles for management, and risk stratification of HF in children and young adults.This study was reviewed and approved by the Indiana University School of Medicine Institutional Review Board and conformed to the declaration of Helsinki. All subjects gave written informed consent if older than 18 year, or assent if younger than 18 year , to participate in the study. From December 2018 to November 2019, children and young adults with SV were recruited from the pediatric cardiology clinics and inpatient cardiology service at Riley Hospital for Children, at Indiana University School of Medicine, Indianapolis, IN, USA. These subjects all had single ventricle hearts of either left ventricle, or right ventricle dominant anatomy, and all had undergone total cavopulmonary anastomosis. Of the 16 single ventricle subjects, 69% (11) self-identified as Caucasian race, 19% (3) as Black race, and 12% (2) as Hispanic race. Children and young adults without heart disease served as controls (Con) and were recruited from the pediatric cardiology clinics. Inclusion criteria for Con subjects included structurally normal hearts. The Con subjects were all healthy and did not have any systemic disease or other heart disease. Of the 10 Con subjects, 100% (10) self-identified as Caucasian race. Management of subjects with SV was based on the standard of care (SOC), and the diagnosis of heart failure was based on clinical judgement of the attending cardiologist for that subject. Exclusion criteria for both cohorts were the absence of any other metabolic or systemic diseases, such as diabetes, cystic fibrosis, inflammation or infection, or cancer, and must not be pregnant.Clinical data was abstracted from the medical records after consent was obtained. Subjects were not fasted overnight prior to blood draw. Between 5 and 10 ml of venous blood was drawn into EDTA anticoagulant tubes from subjects in a sitting position. Bloods were immediately spun at room temperature (20 °C) for 10 min at 2500 × g to separate cellular blood elements from plasma, and the plasma was aliquoted and stored at − 80 °C until analysis. Samples were transported on dry ice for analyses.. Briefly, 100 μl of serum was combined with 300 μl of deuterated phosphate buffered saline (PBS) and the solution was filtered through a 10KDa molecular weight cutoff filter. The filtrate was then combined with deuterated PBS containing contain 2,2-dimethyl-2-silapentane-5-sulfonate-d6 (DSS-d6) to a final volume of 600 ul and 0.5 mM DSS-d6. Spectra were acquired on a Bruker AVANCE III, 700 MHz NMR equipped with a cryogenically-cooled probe. Data was acquired using the 1D NOESY pulse sequence covering 12 ppm, digitized with 32,768 points during a 3.9 s acquisition time. The mixing time was set to 100 ms and the relaxation delay between scans was set to 2.0 s. The data was processed and quantitative spectral fitting of 26 metabolites was carried out using the Chenomx NMR Processor and Profilers software packages  with DSS-d6 as the chemical shift and quantitation standard.Samples for NMR analyses were prepared using established protocols . This is a commercially available targeted metabolomics assay that can be used on a variety of LC/MS/MS instruments. Preparation of serum samples followed vendor protocols. In brief, 10 µL of the supplied internal standard solution were added to each well (except for the zero sample) on a filterspot of the 96-well extraction plate. After drying under a gentle stream of nitrogen 10 µL of each serum sample, quality control (QC) samples, blank, zero sample, or calibration standard were added to the appropriate wells. This assay targeted 629 metabolites across a wide range of chemical classes including amino acids and amino acid related compounds, bile acids, free fatty acids, phosphatidylcholines, sphingomyelins, ceramides, cholesterol esters, diglycerides and triglycerides. Isotope labeled standards for a subset of these metabolites are included in this assay to support quantitation. A subset of 134 metabolite had more than 50% zeros and were therefore not included in the analysis and thus, at total of 495 metabolites were included in the analyses As set of eight QC samples were prepared from pooled sample and included in the run. Data processing was carried out using the Biocrates MetIDQ software. The results of both the NMR and MS platforms were reported in micromolar units. Supplementary Table 1 contains all of the metabolites.www.viime.org)  analysis was carried out in MetaboAnalyst (version 5.0) using the multivariate exploratory analysis functions. Briefly, ROC curves were generated by Monte-Carlo cross-validation with classification and ranking carried out by Partial Least Squares Discriminant Analysis (PLS-DA) using two latent variables.Twenty-six patients provided written consent and were enrolled in the study. Subjects were not fasted for this study. Ten patients had no heart disease and were enrolled in the Con group. Sixteen patients had a diagnosis of SV of which 8 had a diagnosis of HLHS, and 8 had a diagnosis of other forms of SV including double inlet left ventricle, tricuspid atresia, unbalanced atrioventricular canal, and double outlet right ventricle. Of those 16 patients with SV, 9 were diagnosed with congestive HF based on physical examination and required diuretic support. This cohort was designated as SVHF. Seven single ventricle patients did not have a diagnosis of HF, were not on diuretic support, and were designated as SV. The baseline characteristics for each of the three groups are shown in Table There were no significant differences in age, sex, height, or weight between all 3 groups. In addition, there were no significant differences in the anatomic diagnosis, age at surgery, or type of surgical palliation between the SV and SVHF cohorts. The mean oxygen saturation was not different between SV groups (92.9% vs. 92.1%). Cardiac troponin I (cTnI) and C-reactive protein (CRP) were measured in 5 of 7 SV subjects, and 8 of 9 SVHF subjects. The CRP was normal in all subjects except for one child (2.2 years) at 2.9 mg/dL, and the cTnI was normal at < 0.03 ng/mL in all subjects measured. Medication use between the cohorts was not different except for diuretic use in the SVHF group (p < 0.0001). Within the SVHF cohort, 3 had protein losing enteropathy (PLE), and 3 required heart transplantation because of severe HF at a later date. In contrast, no SV patient was being treated with diuretics, had PLE, or required heart transplantation.Integration of the NMR and MS data revealed a set of 44 metabolites for which there was a significant difference in at least one of the inter-group comparisons. Of these significant metabolites, 41 were detected by MS and 3 were detected by NMR. Supplementary Table 1 contains the ANOVA derived p-values for all of the metabolites detected by both platforms. When a metabolite was detected by both platforms, only the MS was listed. A heatmap of the significantly altered metabolites is shown in Fig. A set of 4 acylcarnitines (ACs) were found to be significantly altered, including dicarboxylated (DC) and hydroxylated (OH) species. The short chain, methylmalonylcarnitine (C3-DC-M) was slightly reduced in SVHF compared to both controls and SV. The medium chain pimeloylcarnitine (C7.DC) was increased in both SV and SVHF compared with controls. Trends toward reduced levels of these short and medium chain ACs were observed in SVHF versus SV. The longer chain dodecanedioylcarnitine (C12-DC) was reduced in both SV and SVHF, and the hydroxyhexadecanoylcarnitine (C16-OH) was significantly reduced only in the SVHF group. For these longer chain ACs no significant difference was observed between SV and SVHF cohorts.A set of 8 amino acid-related compounds were significantly altered. Of these, four were canonical amino acids: aspartate (Asp), glutamate (Glu), histidine (His) and threonine (Thr). The significant changes included reductions in both SV and SVHF compared with controls. The SVHF patients were characterized by increased levels of Asp and Glu compared with SV.The four non-standard amino acids include nitro-tyrosine, ornithine, α-aminobutyric acid (AABA) and γ-aminobutyric acid (GABA). AABA was significantly reduced in SVHF and trended downward in comparing SVHF with SV. In contrast, GABA was significantly increased in SVHF compared to both Con and SV. A significant reduction in nitro-tyrosine is only observed in the Con versus SV comparison along with a trending reduction in SVHF compared with SV. Ornithine demonstrated a significant increase in SVHF compared with Con.Detailed analysis of bile acids in young Fontan patients has not been reported before now, nor related to HF status. A set of five bile acids were found to be significantly altered including the secondary bile acid glycocholic acid (GCA) and the conjugated primary bile acids glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GUDCA), taurocholic acid (TCA) and taurochenodeoxycholic acid (TCDCA). All alterations involve increases with a trending increase in GCA in the SV group while four of the five are significantly elevated in the SVHF group. Strikingly, three bile acids, TCA, GCA, and TCDCA, showed significant increases which clearly distinguished the SVHF patients from the SV cohort.Alterations in lipids are represented by changes in 21 metabolites with 16 of these being triacylglycerols (TGs). Interestingly, there were no changes in the TGs in the SV group when compared with the Con cohort, but 13 of these were significantly increased in the SVHF group compared with Con. A set of 5 significant increases in TGs were observed in the SVHF group compared with SV, along with trending increases in 6 others.Additional metabolic changes were found which suggest potential effects on hormone signaling, energy metabolism, and one-carbon metabolism. These have not been described before now in the child with SV. A significant decrease in the androstane steroid, dehydroepiandrosterone sulfate (DHEAS) was observed between Con versus SVHF which persisted when comparing SV versus SVHF. Additional changes were observed in 3-indoleacetate and hippurate. The Krebs cycle intermediates citrate and succinate were both increased in Con versus SV, whereas only the increase in citrate was observed in the Con versus SVHF. Neither Krebs cycle intermediate was different between SV and SVHF. Betaine functions mainly as a methyl donor that plays a significant role in a number of processes including liver function. Significant increases in betaine were observed in the Con versus SV, and Con versus SVHF comparisons.To evaluate the potential of the metabolite profiles as a diagnostic panel, receiver operating characteristic curves were generated using partial least squares discriminant analysis models. Figure The metabolic profiles found in this pilot study significantly expand upon the limited metabolomic studies of SV subjects. Furthermore, these findings demonstrate that: (1) specific metabolic profiles are associated with HF in these patients, (2) these profiles can distinguish between CON, SV, and SVHF and, (3) are found in early childhood. Multiple studies evaluating HF in the SV subject to this point have relied on clinical laboratory data, such as BNP, or multiplex immunoassays, and have advanced our understanding of inflammation and tissue responses at a protein level for the Fontan subject  profiles were found in the study of adult SV patients by Michel et al. In that study, significant elevations in C0 (carnitine), C3 (propionylcarnitine) and C18:2 (octadecadienylcarnitine) were found in the SV patients. In contrast, we found perturbations only in dicarboxylated and hydroxylated species. Moreover, three of the four species were decreased while only one was increased; see Fig. Perturbations to ACs and specifically the dicarboxylated ACs, have been found in several studies of cardiovascular disease. In a metabolomics analysis of serum from 314 individuals with CAD, a principal components analysis derived metabolite factor composed of small to medium chain dicarboxylated ACs significantly predicted an incidence of myocardial infarction (MI) and death (Shah et al., Amino acids are important as biosynthetic substrates for cellular structures and signaling molecules as well as being a source of energy. The circulating levels of amino acids are achieved in part by maintaining a balance between protein synthesis and degradation along with amino acid catabolism. Consistent with the study from Michel et al. we find decreases in Asp, His and Thr in the SV patients, and an increase in Glu in the SVHF compared with SV (Michel et al., The alterations in BAs are an intriguing finding that likely relate to the congestive hepatopathy associated with the Fontan circuit (Emamaullee et al., Bile acid metabolism is also affected by gut microbiota and is known to alter metabolism of the host (Wahlstrom et al., A significant pattern of increased triglycerides was observed in the SV and SVHF group suggesting that dyslipidemia may be a factor in SV heart failure. Michel et al. reported alterations in phospholipid metabolism as evidenced by reduced levels of phosphocholines and sphingomyelins. The increase in triglycerides found in our study may be associated with the alteration in lipolytic processes that can play a significant role in the development of adult heart failures (Kintscher et al., Whitesides et al. reported significant reductions in circulating cholesterol including reductions in total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in a cohort of pediatric SV patients who underwent Fontan palliation (Whiteside et al., Although our current study provides new insights into the metabolic pathophysiology of HF in the child with SV heart disease, there are several limitations. First, this is considered a small study consisting of 10 controls and 16 SV subjects. Single ventricle heart disease is rare and occurs in about 5 of every 10,000 live births and thus, recruiting pediatric subjects with a range of disease states is expected to be difficult (Hoffman & Kaplan, The expanded analyte profiles described here in children with SV hearts are novel and will allow further, hypothesis-driven studies into the metabolic perturbations of the SV patient. Furthermore, there are very clear metabolite differences between SV and SVHF subjects, which may allow for development of these analytes as biomarkers predictive of progression to HF, as well as response to therapeutic intervention. Future studies will need to involve cross-disciplinary expertise and techniques to understand organ dysfunction in the unique physiology of the SV Fontan heart.


The properties of printing for 11 color spots are evaluated by x-rite before and after the cold storage period without any detectable changes in the rate of color change (ΔE). The bacterial count of the tested sample is examined by counting the microbial colonies in the agar plate media. There is good agreement between microbial count and smart indicator color change as an effective direct detection tool for sustainable food quality and safety.Smart packaging, also known as intelligent packaging, is responsive to external stimuli, moisture, light, oxygen, heat, pH, and bacterial growth. It has evolved from extensive applications in food safety, bacterial response, and medical packaging. Interactive packaging has a scientific basis for additional information about food products because these codes give all required data. This work deals with a combination of frontline food sciences, smart and interactive packaging that are applicable for future production of nutrition packages through smart detection of food spooling. Additionally, it verifies the best degree of food safety and population demands as the third generation of packaging technology. High qualified duplex laminated package with a nano-encapsulated pH monitoring label for fresh fish was printed. The interactive Quick Response code icon was combined in a designed package with important information about cooking, smart packaging, and fish quality. Therefore, particle size, zeta potential, and surface area are measured for a nanoencapsulated indicator which exhibits 74.4 nm, 23.6 mV, and 88.9 m Human health has been influenced by the quality of foods and their types. The demands of high food quality products with improved quality and shelf life are on the rise globally. For that, packaging materials play a vital role in saving the food quality, especially the third generation of packaging that indicates the spoiling metabolism of food throughout using smart, responsive materials.In addition, Packaging functions can be classified into the following protection, communication, containment, and convenience . The secSpontaneously, the interactive packaging design based on Quick Response code (QR) adds another dimension to clarify the utilization of smart packaging plus checking the product shelf life. In this kind of interactive smart packaging, a shrewd outsider gadget, for example, a smartphone, is allocated to examine a coding framework to perceive pictures –8 and itFreshness indicators could give quick product quality data because of microbial development or synthetic changes inside a food product . EncapsuMoreover, microbiological quality might be resolved visually through responses between microbial development metabolites and incorporated indicators and pointers inside the package . Most ofThis paper, a smart and interactive package as the third generation of packaging, was designed and printed with a novel nanoencapsulated pH indicator as a color response label. The reverse printing is performed and applied on polyester/polyethylene duplex laminated structure material (PET 12/ PE 40 micron) using a polyurethane adhesive. The microbial population was followed over 288 h and compared with label color transformation as a direct tool for food quality by bacterial population detection. Combining multidiscipline frontier sciences, nanotechnology, smart packaging, and interactive packages has been enhanced for food quality detection to one of abundance food types. The fresh fish filet was kept in storage for over 12 days at 4°C with follow-up from microbial growth and pH response using a validation indicator.The interactive package design was constructed in the Adobe Illustrator CC 2018 program in cyan, magenta, yellow, and black (CMYK) colors with spot colors. Processing machine photopolymer flexo-plate ESKO was used to produce the flexo photopolymer plate thickness 1.14 mm, Germany. The interactive package was printed on flexographic machine supplier Bobst 8 color 2019, Germany. The reverse printing was applied on polyester/polyethylene duplex laminated structure material (PET 12 μm/PE 40 μm) with polyurethane solvent-less adhesive. The rate of color change ΔE was measured with X-Rite exact, Pantone, the USA for the printed packages.Lactic acid, stannous octanoates, and methyl red are purchased from Sigma-Aldrich, Germany. Additionally, chloroform, acetone, and ethanol were distilled and kept over calcium hydrate until used. Nile perch fishes were purchased from the Egyptian food market (Cairo) and were prepared as a filet with direct packaging in a printed duplex smart and interactive package. The nutrient agar  was used, and the potato dextrose agar  was purchased.−1). The 20 ml of methyl red solutions was mixed with (0.21 mol) lactic acid monomer. The mixture was charged in a three-neck round bottom flask connected with vacuum and nitrogen inlets. The reaction flask was immersed in a temperature-controlled water bath during nitrogen purge for 1 h with stirring 800 rpm. The temperature was raised gradually up to 75°C. The polymerization initiator 2.1 × 10−2 mol of stannous octanoates was added dropwise over 15 min. The reaction was continued for 10 h under vacuum. After that, the reaction was immersed in an ice bath, and the prepared methyl red/polymer was precipitated in acetone through a dropwise separation funnel. The precipitate was dried under vacuum at 50°C for 24 h. After dying, the indicator circle was cast in a circle Teflon plate and drying in a vacuum oven at 40°C for 24 h.According to our previous work, with little modification, the synthesis of polylactic acid and encapsulation reaction of methyl red occurred with little modification . Methyl The prepared polymer/methyl red nanoencapsulation was dissolved in chloroform with solid content 20 wt% and mixed for 30 min over a magnetic stirrer. Then, PLA/pH indicator solution was purged with nitrogen for 30 min and immersed in a sonication bath for 10 min to remove any air bubble. The solution was cast in a Teflon Petri dish and dried in a vacuum oven overnight at 40°C. The film was cut into small round pieces to be used as indicator pads. All pads were attached to the inner side of the duplex package film by pressing under a mechanical hydrolytic piston at 80°C for 2 min.4 Quantachrome Instruments, Florida, USA, to determine the Brunauer–Emmett–Teller (BET) surface area. All samples were kept dry in a desiccator until testing. Samples were cooled with liquid nitrogen and analyzed by measuring the volume of gas (N2) adsorbed at specific pressures. The pore volume was taken from the adsorption branch of the isotherm at P/Po = 0.995, assuming complete surface saturation.The mean diameter and zeta potential of the nanoencapsulated particles were determined at 170 and 18°, respectively, by dynamic light scattering (DLS) . Nitrogen adsorption-desorption measurements were carried out at 77.35 K on a Nova Touch LXvia the cup method. The water vapor permeability was measured as the amount of water vapor passes through the tested film. Also, WVTR was measured as the mass of water vapor transmitted throughout a unit area in a unit time under controlled conditions of temperature (38°C) and  according to the following standards . Also, the oxygen transmission rate (OTR) under conditions (23°C and 50% RH) was measured by N530 Gas Permeability Analyzer  according to the following Standards ISO2556-2001, GB/T1038-2000, ASTM D1434-82(2003).Water vapor transmission rate (WVTR) was performed using GBI W303 (B) Water Vapor Permeability Analyzer  2). OM results were calculated according to (EN 1186-5-single side contact in cell test)  Regulation are categorized in Nr. 10/2011 (The PIM), Simulant A, Simulant B, Simulant D2. All samples were compared with a blank sample (Millipore water with resistivity 18.5 MΩ) as a reference. The OM is expressed as the amount in milligrams of material lost from one decimeter square surface (mg/dmll test) .−2–10−6) were performed, and dilutions were plated in duplicate. The spread plate and pour plate methods enumerated total aerobic bacteria (at 37°C for 2 days). In comparison, yeasts and molds (at 28°C for 3 days) were performed on the nutrient agar  and the potato dextrose agar , respectively. The microbial colonies were counted and expressed as CFU/g of the tested sample . Mixed sample with sterilized bi-distilled water (0.1 g/ml w/v) and then transferred aseptically to a stomacher bag and shaking for 4 h at 180 rpm. Serial dilutions /polyethylene (PE) film was reversely printed through the flexographic process and laminated as a high barrier interactive package with excellent durability, environmental stability, and mechanical flexibility characters of used nitrocellulose inks . The PETThe rate change of colors (ΔE) for the printed package before and after cool storage for 12 days at 4°C. Eleven color spots were selected to be measured with X-Rite, as shown in Differences in ΔE (Δ1.10–Δ0.29) for color spots before and after cool storage were tiny. The higher and lower values of Δ1.10 and Δ0.29 were related to color 2 (orange) and color 4 (red). All measurements are within the permissible limits according to ISO 12647-6. The colors of the printing package were not affected through cool storage as customer demand. This is verified the aim of reverse printing technology to protect the color of the printing area .The barrier properties of laminated packaging film were evaluated against oxygen gas and water vapor at 23°C as permeability for the film sector. According to Robertson , the tra−2 day−1 Pa−1 at a humidity of 50%. In addition, the water vapor transmission rate was exhibited high barrier properties for water vapors as a successful anti-moisture packaging film. The measured values are 0.351–2.072 g m m−2 s−1 Pa−1 at water vapor saturation of 80%. The water permeability in this study is lower than PE and PET films in the literature which can be discussed to increase the hydrophobicity of the laminated film with an adhesive layer between the two films (The oxygen transmission rate (OTR) shows excellent barrier property with low oxygen gas permeability with a range of 0.047–0.252 mL m mwo films .Laminated polymer films are materials consisting of multiple layers of different polymers types. These materials are ubiquitous in daily life, with substantial food and pharmaceutical packaging containing laminates. A common requirement of food packaging is that the internal layer must appropriate for contacting food, and the external layer must be suitable for printing product information.2 with limits of accepted level up to 10 mg/dm2, as shown in The migration of low molecular weight polymer or chemical additives substances from the bulk polymers laminated film has been studied according to the EU Regulation Nr. 10/2011. The stimulants were selected carefully to represent the different food natures. As shown in Photobacterium phosphoreum are found in spoiled packed cod suggesting this organism could be responsible for spoilage  with off-odor detection was used as qualitative measurements of microbial spoilage activity . On averspoilage . A high cked cod .−7 and 10−8 after 10 and 12 days, respectively. As a rule, fish spoil more rapidly than meat under chill conditions . The total aerobic bacteria, the total yeast, and molds were reached the lower value even after 96 h, which became neglected compared with the unpackaged sample. After 6 days of storage, yeasts, molds, and total aerobic bacteria counts were initially increased. It was observed that the total yeast and molds were remarkably lower than that observed in the total aerobic bacteria along with the storage time. Indeed, the packaging of fresh fish in the smart package showed the highest preserved against microbial population −7 and 10−8 dilution. Also, the microbial growth for bacteria, molds, and fungi was directly related to pH change due to the metabolism process for fresh fish filets. Overall, effective microbial spoiling metabolism was well directly detected through a smart and interactive package with nanoencapsulated color change pH sensors from yellow to red as an efficient motoring tool to verify the validity of fresh fish to use or discard.Excellent microbial growth detection was monitored through a developed nanoencapsulation technique for pH sensor materials to evaluate fresh fish quality tools. This research-based one combined multidiscipline science, packaging design as an interactive package, nanomaterials stabilization in polymer matrix through encapsulation process, and microbial evaluation for food quality until spoiling. Package design was successfully printed and combined with a nanopolymer-encapsulated methyl red indicator label. The interactive design provided additional value to the user as it facilitated the monitoring of the fresh fish shelf-life. Microbial population study was concluded with the superb microbial growth detection of packaged fresh fish in pH change until 10The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.