Oryza sativa), maize (Zea mays), barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development.The Poaceae, or grasses, include many agriculturally important cereal crops such as rice ( AbioticReproductive organs are significantly more vulnerable to high temperatures than other plant organs. Planting of crops is typically timed to minimise high temperature exposure during the later reproductive stages, however the fluctuations and extremes of temperature that are now occurring mean that there is an increasing probability that peaks of high temperature will overlap with the flowering period . It is pPrior to fertilisation, One challenge of studying the response of florets to heat stress in cereals is determining when and where defects appear, how this differs between cultivars with different phenology and architecture, and how this might be examined in a non-destructive manner. In this study we analysed the effects of heat stress during floret development in three European spring barley varieties under controlled environmental conditions. One of the varieties, RGT Planet, represents a variety of considerable promise for high yield and malt quality in diverse conditions, including dryland environments in Australia . Our ove22.1Hordeum vulgare; Optic (derived from the cross Chad × (Corniche × Force), RAGT Moonshine and RGT Planet) were provided by the Wilson lab (University of Nottingham) and were sown in John Innes Potting Compost No.3 in 13cm diameter pots for germination and early plant establishment. All three are elite lines that have been on the AHDB Recommended List, although RAGT Moonshine and Optic were removed from the AHDB Recommended List in 2016  compost in 13cm diameter pots. Heat stress was applied to two stages of reproductive development  Pollen Mother Cell (PMC) meiosis heat stress, 2) Pollen mitosis heat stress and 3) control. The periods of heat stress were carried out in a controlled environment growth room with a day/night temperature of 30/25°C, 80% relative humidity and a 16/8 -hour light/dark photoperiod to mimic rapid onset heat stress conditions. The temperatures were chosen to mimic heat waves occurring more frequently in Europe. Tillers were selected at the beginning of meiosis or mitosis, and were tagged for analysis before being submitted to heat stress. All pots were monitored daily and watered to avoid concurrent drought stress. Plants undergoing the PMC meiosis heat treatment were submitted to heat stress for two days, while plants undergoing the Pollen mitosis heat treatment remained in the heat stress conditions for five days to ensure completion of pollen mitosis I and II, before being transferred back to control conditions. Control plants were maintained at a constant 17°C, 80% relative humidity with a 16/8 -hour light/dark photoperiod.2.2Pollen viability in control and heat stressed plants was assessed by analysis of starch levels in pollen grains using 0.2% (w/v) potassium iodide and 1% (w/v) iodine to determine their ability to germinate and fertilize . Normal 2.3® 488 IgG (H+L)  was used for BG1 and Dylight 550 Goat anti-rat IgM  was used for LM19. The slides were incubated for 2h and then washed with Incubation buffer. 0.1% (w/v) Calcofluor white was added and washed off before imaging with the Zeiss Axio Imager 2 as described in Florets were harvested from the tillers tagged for heat stress during pollen meiosis immediately before anthesis, fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) with 0.1% (v/v) Triton X-100 and 0.1% (v/v) Tween 20 and embedded in paraffin. Floret sections (8μm) were mounted on slides at 42°C, the paraffin was removed from the sections using 100% Histoclear (v/v) and tissue was rehydrated by using an ethanol series ) and water. Slides were washed with 1xPBS and treated subsequently with glycine to inactivate residual aldehyde groups. They were then washed with Incubation buffer (1% (w/v) bovine serum albumin (BSA)) in 1xPBS . Primary2.4The Phoenix Nanotom S, a nanofocus Computed Tomography (CT) system was used to scan barley florets of the three varieties at the Hounsfield Facility in Nottingham as previously described  and AtHSP70 (AT3G12580.1) a nucleotide BLAST search was carried out in NCBI, rice database and IPK Barlex database. Orthologs were identified for rice  and barley  and verified by multiple alignment through phylogenetic tree analysis (http://www.geneious.com). These proteins were chosen to give an indication of differences between varieties in the response of heat shock proteins to heat stress. Due to the high similarity between the proteins in the cytosol subgroup of the HSP70 family, we focussed only on the closest orthologues in barley.To determine orthologs of 2.8® Plant Mini Kit  from at least five florets and pooled together. cDNA was synthesised from total RNA using the SuperScript™ III reverse transcriptase . qRT-PCR was performed with primers for HvHSP17.8 (HvHSP17.8 Fw 5’GAGGTGGAGGACGGCAACA3’ and HvHSP17.8 Rv 5’GATGGACTTGATCTCGGGTTT3’) and HvHSP70 (HvHSP70 Fw 5’ATCCTGAACGTGTCTGCTGA3’ and HvHSP70 Rv 5’TGGTGTTGCGCATGTTGTAA 3’) on three technical replicates and analysed using the LightCycler® 480 . Expression values relative to the barley α-Tubulin gene (HvTubF 5’AGTGTCCTGTCCACCCACTC3’ and HvTubR 5’AGCATGAAGTGGATCCTTGG3’) were calculated.RNA was extracted with the RNeasy33.13  in Optic and 1.1 mm3  in RGT Planet. Whereas the PMC meiosis heat stressed anthers were smaller: 0.29 mm3  in Optic and 0.71 mm3  in RGT Planet  that had been exposed to heat treatment at PMC meiosis and Pollen mitosis, and compared to those from the control conditions. Staging of anther development was conducted according to the non-destructive reproductive staging approach of T Planet Figure 2Immunolabelling was subsequently used to observe sections of the anthers and highlight cell wall components such as 1,3;1,4-β-glucan, de-esterified homogalacturonan and cellulose to determine if the cell wall composition was impacted as a consequence of the heat stress , which is smaller than the heat stressed carpel, which measured 2.58mm3 . The X-ray imaging also showed a significantly enlarged carpel in the RGT Planet heat-stressed floret with a volume of 4.46mm3  compared to the control which had a volume of 1.37mm3   Figure 2The immunolabelling of ovule sections in Optic only showed slight differences between control and heat stressed florets  involved in protein stability, while HvHSP70 is a chaperone involved in protein folding  of pollen mitosis heat stress for all three varieties and control plants  showed severe morphological deficiencies in anther development, and these are likely to be a leading cause of sterility due to high temperature stress. A short period of heat stress during early floret development resulted in significantly smaller and deformed anthers. Although the anthers were harvested at the same time from heat stressed and control plants, the septa in the heat stressed anthers had already broken, suggesting a more rapid developmental progression and/or a change in the physical properties of the anther walls. It has been suggested that thick locule walls and well-developed cavities in the septa may be responsible for heat tolerance in rice . The cavin vivo after heat stress.In addition to defects in anther morphology, pollen development was severely affected by heat stress during both PMC meiosis and pollen mitosis, with decreased viability, pollen count, and accumulation of starch. However, as shown by the X-ray micro CT images of Moonshine florets after PMC meiosis heat stress, and by the presence of viable pollen in Optic after meiosis heat stress, pollen is still formed in some heat-stressed florets and, based on KI staining and seed counts, some may be viable. The absence of pollen detected in anthers during immunolabelling may be due to early anther rupture and loss of pollen during fixation and embedding. Alternatively, many florets may lack pollen entirely, and pollen is only released from some florets that avoid stress based on their position within the spike and their stage of development, which varies between the central and terminal regions. This highlights the importance of optimising non-destructive X-ray CT to assess anther development along the spike Despite apparent contradictions between the immunolabelling experiments, KI staining assays, and CT data in regard to pollen grain number and viability after heat stress, seed set was used as a functional output of floret fertility. This varied between the treatments and cultivars. In Moonshine, overall seed set was significantly reduced across many tillers after heat treatment at PMC meiosis or pollen mitosis, but analysis of individual spikes suggested that Moonshine was more resilient against heat stress at the pollen mitosis stage. Optic, on the other hand, showed no significant response to heat stress at PMC meiosis, but was strongly sensitive at pollen mitosis. In contrast, RGT Planet was sensitive to heat stress at both PMC meiosis and mitosis stages of pollen development. These results potentially reveal different temporal sensitivities to heat stress in three elite barley cultivars.The reduction in floret fertility and seed number may result from multiple defects in the pollen after heat stress. This includes reduced starch accumulation, which was observed in Optic pollen after heat stress at pollen mitosis. It has been suggested that stresses such as water stress can inhibit starch deposition in rice and wheat pollen, either by decreasing the availability of assimilates or by impairing the activities of enzymes involved in starch biosynthesis . PressmaThe female reproductive organs did not show any significant differences in embryo sac phenotypes after heat stress. However, the carpels were bigger, possibly as a result of swelling of the unfertilized ovaries similar to that observed in wheat . MoreoveSeveral studies indicate that both pre- and post-fertilisation stages of ovary development are sensitive to stress, but this varies depending on the species and genotype . In wheaHvHSP17.8 and HvHSP70, were upregulated after heat stress. HvHSP17.8 is a small heat shock protein (sHSP) that assists in preventing aggregation and stabilizing proteins while HvHSP70 is a chaperone that assists in protein folding processes  showed more severe effects in the male reproductive organs in Optic than in the other two varieties, with no viable pollen and significantly more sterility. The female reproductive organs of the three cultivars showed no severe effects after heat stress, at least in terms of embryo sac expansion. Overall, RGT Planet performed worst compared to the control conditions, whilst Moonshine performed best. However, there were less florets per spike in the heat stressed plants across all three varieties compared to control plants. Further investigation will be required to identify the traits that contribute to Moonshine being more heat tolerant than the other two varieties. The data provided here provides a basis for further studies into differential heat stress responses during key stages of barley reproductive development. Moreover, the use of X-ray imaging is a promising new method to visualise the morphology of floret organs without dissection and manipulation with chemicals, giving a more representative image and accurate volume measurements. Further work is needed to determine how the impact of the heat stress observed here equates to that under field conditions and the resultant potential consequences for yield loss. Nevertheless, significant yield reductions have been observed , so it sThe original contributions presented in the study are included in the article/CC, ZW, and DZ conceived the study. CC carried out the majority of experiments and data analysis. JFG assisted with plant maintenance and spike analysis. MT assisted with immunolabelling. All authors contributed to the article and approved the submitted version.


The DPPH radical scavenging activity and ORAC value of soybeans  were increased by roasting at above 190 °C. Concerning raw beans, black soybeans with the darkest seed coat color had the strongest antioxidant activity, indicating the effect of the coat pigment. However, the degree of increased antioxidant activity by roasting was almost similar regardless of seed coat color, suggesting that coat color is independent of the increased antioxidant activity. Concerning aged beans stored at 37 °C/75% RH for 60 days, the antioxidant activity increased in yellow soybean and decreased in blue and black soybean compared to before storage. Conversely, when roasted at 190 °C for 20 min, the DPPH values of all the aged beans were significantly increased. Other analyses of roasted beans with and without seed coat showed that changes in the components of cotyledons during storage may have contributed to the increased antioxidant activity of aged beans, regardless of seed coat color. These results revealed that roasting effectively improves the antioxidant activity of aged soybeans, regardless of seed coat color. We concluded that roasting is recommended for antioxidant properties, particularly regarding the effective use of aged beans. Soybeans are important foods because they retain high-quality nutrients and various functional components, such as polyphenols. The polyphenols in soybeans are mainly isoflavones with antioxidant properties. The seed coat of beans, which protects the cotyledon from the natural environment, contains many polyphenols that suppress oxidative damage in the cotyledon and the human body ,2. VarioCommon beans stored in adverse environments, such as high temperatures and humidity, are less likely to soften, even when cooked for a long time. This phenomenon is called the “hardening” or “hard-to-cook (HTC) defect”, which has been a serious problem in many countries where beans are staple foods ,5. The cIn food processing, soybeans are sometimes roasted, ground into flour, and used as soybean flour with a good flavor. This flavor originates from the Maillard reaction products generated during food heating with high antioxidant activity. In various foods, such as sesame seeds, cashew nuts, and corn, the browning substances generated from the Maillard reaction are reportedly produced by roasting, with the antioxidant property increasing ,12,13. TGlycine max L. Merrill) with different seed coat colors were used as samples. Yellow , blue , and black soybeans , harvested in 2019, were purchased from an online bean store. The general composition of soybeans with different seed coat colors was similar . The samples were heated at 135 °C for 4 h and weighed until they reached a constant weight. The weight loss of the samples corresponded to the water content; the dry weight of each sample was determined.The color values were analyzed using a spectrophotometer  represented by L*, a*, and b* according to the CIE color scale. L* represents the brightness from 0 (black) to 100 (white); the other two coordinates represent redness (+a*) to greenness (−a*) and yellowness (+b*) to blueness (−b*), respectively. The experiment was repeated three times. The hue change (ΔE) due to roasting was calculated using the following formula:Samples used for the component analysis were ground into flour (Kawasaki Crusher DM-6) and passed through a No. 30 mesh screen. These processes were performed in a controlled room (4 °C) to avoid frictional heat effects. Approximately 1 g of soybean powder was added to 50 mL of 80% ethanol or 80% methanol solution and was shaken at room temperature . The mixture was centrifuged ; the supernatant was filtered through a 0.45 μm membrane filter. The extracts were used to analyze the antioxidant activity and total phenol content.Extracts obtained by 80% ethanol were used to analyze 1,1-diphenyl-2-pucrylhydrazy (DPPH) free radical scavenging activity and Oxygen Radical Absorbance capacity (ORAC).DPPH’s free radical scavenging capacity was evaluated using the method described by Blois (1958) , with moThe ORAC assay was performed as described by Cao et al.  and Huanv/v) Na2CO3 (0.2 mL) were mixed. After the mixture was allowed to stand at 26 °C for 60 min, the absorbance was measured at 760 nm. The sample concentration was obtained from a calibration curve using the standard; the results are expressed in mg per gram of dry weight. The experiment was repeated three times.The total phenolic content (TPC) was determined using the Folin–Ciocalteu method, with tannic acid as the standard. The extract obtained by 80% methanol as mentioned above (0.5 mL), distilled water (0.5 mL), 0.1 mL of 4-fold diluted Folin–Ciocalteu’s reagent solution, and 10% (50) was calculated. The experiment was repeated three times.ACE inhibition activity was tested in 80% ethanolic extract. ACE inhibition activity test was conducted using ACE kit-WST . The assay procedure was in accordance with the technical manual provided with the assay kit. Briefly, ACE inhibition activity was tested by pipetting 20 μL sample solutions and 20 μL substrate buffers to the sample wells, then adding 20 μL of enzyme solutions and the mixtures were incubated at 37 °C for 60 min. Absorbance was measured at 450 nm using the microplate reader Synergy HTX . The ACE inhibitory activity of each extract was measured, and the 50% inhibitory concentration (ICp < 0.05) using SPSS Statistics version 25.0 software for Windows .Data are expressed as the mean ± standard deviation. The significance of differences between control and each treatment was analyzed using Student–Newman–Keuls test. Data analysis was subjected to multiple comparison tests (As shown in The changes in DPPH and ORAC values of soybeans with increasing roasting temperature are shown in The TPC of the soybeans before and after roasting is shown in The Maillard reaction is a chemical reaction between nitrogen compounds, such as amino acids, and carbonyl compounds, such as reducing sugars, during the heat processing of food, producing brown substances and distinctive flavors. Melanoidin, a brown pigment formed in the late stages of the Maillard reaction, has reducing and antioxidant properties . FurtherTherefore, we proposed that the increase in antioxidant activity by roasting is related to Maillard reaction products and phenols produced in the cotyledons rather than in the seed coat. In addition, temperatures of ≥190 °C were needed to increase the antioxidant activity. Although this activity was the highest at 230 °C, 190 °C was regarded as the appropriate roasting temperature because the color of roasted soybeans was too dark, as mentioned in The effects of soybean storage and roasting on color values are shown in The effects of storage and roasting on TPC are shown in To investigate the effect of seed coats on antioxidant activity, the seed coats of the roasted beans were removed, and the DPPH values were compared with those of the beans with seed coats. However, the seed coat of the raw beans could not be removed because they were firmly attached to the cotyledon. The results are shown in Conversely, when aged beans were roasted at 190 °C, the DPPH radical scavenging activity of all the aged beans without seed coats was significantly higher than that of those with seed coats. DPPH values are unaffected by removing the seed coat if the compositions related to the antioxidant activity are distributed equally in the seed coat and cotyledon. However, as seen in These results revealed that roasting increases the antioxidant activity of aged beans due to changes in the cotyledon’s components during storage, even if storage caused a decrease in antioxidant activity.50 values indicate that the smaller the value, the higher the ACE inhibitory activity. The IC50 values of aged beans were significantly lower than those of control beans before roasting. Although there was a small decrease in IC50 for both control and aged beans after roasting, the high values of all aged beans were almost maintained. These findings suggest that ACE inhibitory activity increases when soybeans are stored at high temperatures and humidity, regardless of the seed coat color.Finally, we examined the ACE-inhibitory activity of soybeans. Peptides obtained from fermented or enzymatic hydrolysates of soy proteins have been reported to have ACE inhibitory activity . ACE is Betancur-Ancona et al.  reportedBesides the antioxidant properties, an increase in ACE inhibitory activity after soybean storage was observed, and the activity was almost maintained even after roasting. These findings suggest that roasting aged beans are recommended from the viewpoint of nutrients and functional properties.We investigated the effects of storing and roasting soybeans on their antioxidant properties, using aged beans that caused hardening in an HTC state. Three kinds of soybeans with different seed coat colors were roasted at 150–230 °C for 20 min. Consequently, DPPH radical scavenging activity and ORAC value similarly increased at temperatures above 190 °C. Concerning raw beans, black soybeans had the highest antioxidant activity, indicating the effect of pigments in the seed coat. Concerning aged beans stored at 37 °C/75% RH for 60 days, DPPH radical scavenging activity increased in yellow soybeans and decreased in green and black soybeans. Conversely, when roasted at 190 °C for 20 min, the antioxidant activity of all the aged beans significantly increased.The analysis of roasted beans with and without seed coat suggested that changes in some components of the cotyledon during storage contributed to the increased antioxidant activity of aged beans by roasting, regardless of the seed coat color. These results revealed that roasting effectively improves the antioxidant activity of aged beans. In addition, we found that three kinds of soybean showed increased ACE inhibitory activity during stor-age; their values were nearly maintained even after roasting. We concluded that roasting beans is recommended for improved antioxidant activities, particularly regarding the effective use of aged beans.


This research is motivated by the firm believe that these nanomaterials have potential for contributing to the solution of environmental problems and, conversely, will not be part of the problem. Therefore, assessment of the impact of nanoengineered materials on eco-systems is important and research in this area has also been developed by collaborative projects involving experts in nanotoxicity. The above topics are reviewed here by presenting a brief conceptual framework together with illustrative case studies, in some cases with original research results, mainly focusing on the chemistry of the nanomaterials investigated for target applications. Finally, near-future developments in this research area are put in perspective, forecasting realistic solutions for the application of colloidal nanoparticles in water cleaning technologies.Water is the most important resource for all kind forms of live. It is a vital resource distributed unequally across different regions of the globe, with populations already living with water scarcity, a situation that is spreading due to the impact of climate change. The reversal of this tendency and the mitigation of its disastrous consequences is a global challenge posed to Humanity, with the scientific community assuming a major obligation for providing solutions based on scientific knowledge. This article reviews literature concerning the development of nanomaterials for water purification technologies, including collaborative scientific research carried out in our laboratory (nanoLAB@UA) framed by the general activities carried out at the CICECO-Aveiro Institute of Materials. Our research carried out in this specific context has been mainly focused on the synthesis and surface chemical modification of nanomaterials, typically of a colloidal nature, as well as on the evaluation of the relevant properties that arise from the envisaged applications of the materials. As such, the research reviewed here has been guided along three thematic lines: 1) magnetic nanosorbents for water treatment technologies, namely by using biocomposites and graphite-like nanoplatelets; 2) nanocomposites for photocatalysis (e.g., TiO Research on colloidal inorganic nanomaterials has developed considerably in the last decades. Taking the last century transition period as a yearly timeline reference, it is possible to trace to about that period the development of important chemical methods for the synthesis of quantum dots, magnetic metal oxides and metals, just to mention a few relevant classes of nanomaterials . AssociaThe unique properties of inorganic nanoparticles make them suitable for applications in diverse areas, which include nanomedicine  , energy Several water cleaning technologies have been developed, including adsorption, photocatalysis and membrane filtration. Adsorption is an efficient method, very attractive due to the affordable cost and easy operation. Photocatalysis enables water treatment in an eco-friendly manner because solar radiation can be used as an energy source. However, complete mineralization of the pollutants is challenging and may result in oxidation by products formation. The interest in membrane filtration technologies has grown significantly in the last years, but it still faces challenges in membrane fouling, concentrate disposal and high energy consumption. Together with remediation technologies, preventing measures should be also implemented namely by developing water quality monitoring protocols, that can be easily accessed in different regions of the globe. As an example of this approach, several sensors have been investigated, namely envisaging the optical detection of water contaminants using low-cost and disposable substrates. In this work, we focus on the research of colloidal inorganic nanomaterials and their composites, aiming at nanotechnologies for water cleaning and quality monitoring, which has been a common ground for some of the research carried out in our laboratory during the past years.Water and wastewater management is a major global concern considering an ever-increasing population, Human-driven water pollution and the scarcity of freshwater resources. Specifically, water pollution is critical since it may cause negative impacts on ecosystems and Human health, and it can as well adversely impact economic growth and the social perspectives of societies. Considering this framework, the Organization of United Nations (UN) acknowledged that the availability of good water quality is vital for all living creatures and must be considered a worldwide challenge for this century. The preface of the last edition of the “Guidelines for drinking-water quality” published by the World Health Organization begins with the undisputable sentence: “Access to safe drinking-water is essential to health, a basic human right and a component of effective policy for health protection” . Among tIn this context, several nanotechnologies have been developed with potential for implementation and contribute for water remediation and quality improvement. Most of these procedures rely on the use of nanoengineered materials, which have been surface modified accordingly to the envisaged application and the target contaminants to be eliminated. Probably future developments in this area should be better considered as complementary to conventional water cleaning technologies, which for several purposes are quite effective but less so for the elimination of CEC in certain aqueous environments. Also, water quality monitoring technologies that are easily implemented and communicated in different parts of the globe, including in remote regions, are regarded as important advancements, namely because they value prevention behaviour in relation to remediation practices.The following article sections are built upon research lines that converge in providing colloidal nanomaterials for water quality improvement and monitoring. Adsorption techniques applied to water purification are well-established and carbon activated sorbents are ubiquitous in several procedures. However, the removal of certain water pollutants can be ineffective by using such processes, which requires non-conventional approaches. Surface modified nanosorbents for magnetic separation technologies and multifunctional nanostructures for photocatalysis are examples of nanomaterials that might have impact on new technologies for CEC removal or its prevention. Under this scope, chemical gardens have also attracted our attention. These are self-organizing structures created by nonequilibrium processes and have been known since the discoveries made by Johann Glauber (1646). Still, chemical gardens hide new possibilities for technological applications, including selective adsorption-desorption processes. It is known that these micro- or nano-tubules have internal reactive surfaces whose chemistry can be explored to develop sorbents for CEC.Contexts of applications are diverse but typically might include water treatment stations of industries, hospitals, and municipalities. These applications are clearly dependent on the functionality and associated efficacy of the nanoengineered materials, for which surface chemistry plays a major role. The presence of CEC in water, even in vestigial amounts, poses serious threats to human health and jeopardizes the access of populations to safe drinking-water. Thus, not only water purification methods effective for pollutants’ diminutive amounts are required, but accessible and sensitive sensors for water quality monitoring would be a step forward for management of water supplies. Nanotechnology provides a myriad of possibilities for fabricating reliable sensors, which ideally should also be low-cost and user friendly. Together with technologies based on the internet of things, these sensors might offer on-time answers for water contamination events which might occur in remote zones. As it happens for water remediation and purification technologies, the nanosensors’ performance depends on the materials used, namely on their selectivity and nanoscale properties that can be explored for achieving lower detection limits in the field analysis. Optical sensors based on low-cost and versatile SERS substrates are among the most promising for several applications in this context. The risk-benefit balance of these nanomaterials in water cleaning technologies is crucial to decide on their practical use in real contexts. Studies on the assessment of the impact on health and environment of these materials have been carried out, providing a wealth of data that requires a critical and thoroughly analysis. Our research in this area has been mostly collaborative, in providing nanomaterials that have been found as promising for experts on nano- and eco-toxicology to assess, as is also illustrated in this work as an important part of research in this area.Biopolymers, i.e., natural polymers produced by living organisms, are sustainable materials with low toxicity, abundant and available, whose structure contains functional groups with an affinity towards diverse pollutants. The properties of biopolymers and their composites have prompted a focus on water treatment applications, namely as adsorbents for water decontamination . One of 3O4 nanoparticles during the encapsulation process, yielding an organic-inorganic hybrid shell enriched in the biopolymer component. The encapsulation encompasses the hydrolysis and condensation of a mixture of tetraethyl orthosilicate (TEOS), as the SiO2 precursor, and a silicon alkoxide derivative of the biopolymer, in the presence of colloidal magnetic particles. 3O4 nanoparticles. This method originates more robust coatings and with a higher degree of functionalization when compared to the conventional techniques of non-covalent coating and surface-grafting approach, which is desirable to obtain reusable sorbents with improved adsorption capabilities. A series of magnetic biosorbents have been prepared by using this route, from polysaccharides with distinct ionic character and variable chemical functionalities, which were efficient in the uptake of target pharmaceuticals , thereby conferring ability for magnetic separation of the sorbents when exposed to an external magnetic gradient.Our research group has been interested in exploring GNP for their adsorption capability and ability to enhance the adsorption capacity of various composites, with the aim of removing toxic metal ions from water and/or recovering valuable elements, such as technology critical elements. There has been great interest in this topic  because in situ alkaline hydrolysis or electrostatic assembly of particulates previously synthesized. 3O4/GNP and 29 emu/g for the CoFe2O4/GNP and MnFe2O4/GNP composites. As expected, these estimated saturation magnetization values are lower than the values of the respective spinel ferrites at room temperature .The magnetic nanostructures have been prepared by 3O4/GNP nanocomposites were investigated to capture selected lanthanoids , from unary and ternary solutions, and their performance was compared with the one observed for the isolated components (Fe3O4 and GNPs) (3O4 NPs and Fe3O4/GNPs to a ternary solution of La(III), Eu(III) and Tb(III), with an elemental concentration of 100 μg/L, the results show distinct affinity between materials and elements, with the Fe3O4/GNPs composite showing better capability to remove the target elements from water than any of its counterparts (La: 4.7% (GNPs), 3.1% (Fe3O4 NPs), and 15% (Fe3O4/GNPs); Eu: 1.2% (GNPs), 0.2% (Fe3O4 NPs) and 37.6% (Fe3O4/GNPs); Tb: 0.2% (GNPs), 0.1% (Fe3O4 NPs) and 35.0% (Fe3O4/GNPs)). The better performance of the nanocomposites can be explained by an increase of active sorption sites due to the introduction of oxygen moieties into the carbon lattice. Trivalent lanthanides are hard Lewis acids with strong chemical affinity for oxygen donors. More, the sorption process showed to be very dependent from solution pH, and only feasible for pH values higher than 5.2, which is the experimental isoelectric point of the composite (3O4/GNPs) and using 0.1 M HNO3 as eluent solution. During the consecutive cycles, the recovery efficiency of the target elements ranged from 82 to 92% for La (III), 88–104% for Eu (III) and 82–97% for Tb (III). These data elucidate that dilute nitric acid solution (0.1 mol/L HNO3) is an efficient eluent for the recovery of these elements from the magnetic composite. More, the capability of this nanostructure to remove the selected TCE from water was rarely affected since the values of the removal efficiency remained nearly constant during all cycles, also confirming the stability of the magnetic composite.The Fend GNPs) . After aomposite . Consecu3O4/GNPs, CoFe2O4/GNPs and MnFe2O4/GNPs composites were investigated to capture mercury and arsenic, from unary and binary solutions. Among the toxic substances of anthropogenic and natural origin, these elements pose greatest threat to the environment, due to their non-degradable nature, severe toxicity and bioaccumulative character  (1000 μg/L) and Hg(II) (50 μg/L), the results showed that 1) Fe3O4/GNPs were the least effective for removing As(III) or Hg(II) and, 2) As(III) removal was higher for the CoFe2O4/GNPs at pH 7 (90.4%), while MnFe2O4/GNPs displayed higher efficiency to remove Hg(II) at pH 6 (90.6%). In binary solutions, CoFe2O4/GNPs showed more efficiency to As(III) with removal percentages near 90% in ultra-pure water and around 80% in tap water, while MnFe2O4/GNPs removed preferably Hg(II) (85%) in both matrices. Although the above nanocomposites were prepared by similar methods, those containing Co and Mn ferrites performed better than the Fe3O4/GNPs but on the other hand show more potential for metal leaching  region.TiO species . In partlectrons . Previouatalysts . Typicalatalysts . Alternaolutions . There i2 photocatalysts were designed for their magnetic properties through the co-precipitation of magnetite (Fe3O4) in the presence of P25 TiO2. Magnetic water-dispersible TiO2 nanostructures, with a magnetite content of 86 wt% (hybrid A) and 67 wt% (hybrid B), have been prepared by varying the reactants ratio. The ensuing hybrid nanostructures were composed of spheroidal magnetite 12 ± 2 nm in diameter and a TiO2 nanoparticles (24 ± 9 nm), in a ratio 80/20 wt% of anatase/rutile phases. The Fe3O4/TiO2 hybrids were characterized by an absorption edge in the visible region, which is dominated by the Fe3O4 adsorption whose direct band-gap energy is 2.27 eV. The magnetic TiO2 hybrids were then tested for degrading AOX compounds present in the effluent of the bleaching stage from Kraft pulp mills (pH = 2), under UV light irradiation in the presence of H2O2 as the co-catalyst.First, the TiO2 content in the hybrid photocatalyst. Although the highest AOX removal was observed when using non-magnetic TiO2, the magnetic hybrids enabled 59–73% AOX removal after 60 min under UV irradiation  show multi-advantages for application as photocatalysts , namely In this work we have been developing new nanostructured hybrid materials based on polyoxometalate clusters supported in graphene . Graphen11CoO39]5− supported in rGO (rGO-PW11Co), the photodegradation of Rhodamine B (RhB) under visible-light irradiation was conducted at room temperature. RhB was selected as an organic dye model because it shows strong absorption in the visible region  that shows activity under promising conditions, in particular the use of visible-light for irradiation and room temperature.The later example presents a POM based heterogeneous photocatalyst  did not modify the morphology, porosity or the intrinsic hydraulic resistance of ceramic membranes, however, it significantly increased the rejection of humic acid. Higher loading of iron oxides promoted a porous obstruction, thus a drop in permeability.Surface-coated membranes can be obtained by dip-coating, electrospraying, sputter deposition or gas-phase deposition which generates a nanoparticles layer on its surface . HorovitRecently , observe2 nanoparticles in porous PVDF/NaY-based membrane via solvent casting method. Interestingly, the authors reported a minor loss (∼3%) in pollutant removal efficiency compared to the nanoparticle suspension assays. Following a similar strategy, 2 in PVDF membranes with strong antifouling properties under visible light irradiation. The use of blended nanoparticles promoted the degradation of the pollutant while enhancing the filtration performance due to lower membrane fouling. Yet, the membrane permeability can be compromised due to porous hindrance when high nanoparticle loading is used.Matrix-blended membranes obtained by blending nanoparticles in the membrane matrix allow to minimize the possible catalyst leaching. For fabrication, nanoparticles are dispersed by mixing in the polymer-solvent solution before phase inversion. In summary, nanoparticles immobilized in membranes is an effective strategy to overcome the disadvantages associated with conventional slurry-type systems. Surface-coated membranes can improve hydrophilicity and provide antifouling properties; however, the strategy can be ineffective in preventing catalyst leaching. Nanoparticles blended in the membrane matrix are more successful in retaining the catalyst, but the antifouling capacity can be inferior. Deeper optimization to assure porosity, surface dispersion, high photocatalytic activity and improve mass transfer is required to enlarge the broad market commercialization of self-cleaning nanocomposite membranes for environmental application.Fleischmann and co-workers noticed the SERS effect for the first time in 1974 , with itin situ growth of NPs within the filter membrane  coatmembrane ; 4) modimembrane .in situ reduction, immersion-adsorption, and filtration. The latter method provided the SERS substrates with the best stability, reproducibility, and the lowest detection limit for the selected pesticides, compared with those prepared using other methods. Gao and co-workers have developed a rapid and sensitive methodology to detect the fungicide ferbam in aqueous samples using a portable Raman spectrometer  for water CEC monitoring . A simplin situ citrate reduction method applied to Ag(I) dissolved in LCP aqueous suspensions resulted in Ag/LCP fibres, which were then pressed into filter membranes. As a proof of concept, the Ag/LCP filter membranes were supported on polyamide filters, which combine the SERS technique and share similarities with solid phase extraction due to the rapid collection of the molecular analyte for in situ detection  2016/1.Alternatively, the etection . These Setection . These mtert-butyl acrylate)) and used them as SERS substrates  assembled onto surface modified Fe3O4 NPs (ca. 80 nm); the Au NPs are responsible for SERS activity while Fe3O4 allows the separation of the multifunctional particles by applying an external magnetic gradient. The attachment of the Au NPs followed previous surface modification of the Fe3O4 particles with silica shells enriched in dithiocarbamate moieties by using a single-step route  the assembly of colloids of both phases and 2) the ep route . Hence, ep route .3O4-Au nanosorbents in the uptake and SERS detection of water contaminants of emergent concern, such as vestigial pharmaceutics  and biocomposite magnetic nanosorbents. The latter samples have distinct surface chemistry, one sample comprise coatings of k-carrageenan hybrid siliceous shells (Fe3O4@SiO2/SiCRG) and the other contains chitosan hybrid siliceous thin shells (Fe3O4@SiO2/SiCHT). In particular, the selected biocomposite sorbents are of particular interest because they have shown capacity to remove CEC from aqueous media as discussed above , Fe3O4@SiO2/SiCRG (52.4–2000 mg/L) and Fe3O4@SiO2/SiCHT (52.4–2000 mg/L). R. subcapitata (p < 0.05). Regarding Au-NS, the concentration causing 20%  and 50% (EC50) of reduction in the growth rate were 32.3 (95% confidence limits-95%CL: 8.84–7.4) and 294.8 (95%CL: 134.4–455.1 mg/L), respectively. Considering these ecotoxicity results, the safe use of Au NS depends on the amount employed for water quality monitoring applications. For example, in a study on magnetite decorated with Au-NS for SERS detection of tetracycline, only 0.250 mg/L of Au-NS were necessary  mg/L and 164 (95% CL: 131-198) mg/L for Fe3O4@SiO2/SiCRG and of 29.7 (95% CL: 7.55–51.9) and 139 (93.8–183) mg/L for Fe3O4@SiO2/SiCHT, respectively. The ECx values computed for Fe3O4@SiO2/SiCRG were lower than the concentrations used by (3O4@SiO2/SiCRG (by adding 10 mg of the sorbent to 2 ml of a metoprolol solution) to remove metoprolol from water and reported a removal higher than 30% after 2 h contact time. This removal efficiency occurred at concentrations of sorbent that may induce toxicity to freshwater biota, since it is higher than the concentration that impairs in 20 and 50% the growth rate of R. subcapitata. Given that microalgae are at the base of the trophic level, cascade effects at upper trophic levels may be expected to occur. A direct comparison of the ECx here computed for Fe3O4@SiO2/SiCHT could not be done with the amount needed to remove the non-polar compounds  to better understand how the nanosorbents will behave in natural freshwaters. Moreover, scientific evidence exists reporting how environmental characteristics may affect the fate and behaviour of nanoparticles, which, in turn, influences their toxicity to biota [e.g., (The preliminary results on the ecotoxicity of these three systems, suggest the potential for the safe use of Au-NS in the monitoring of water quality. For Fea [e.g.,  and refea [e.g., .This review focused on recent research on nanomaterials of colloidal nature, to be used in this form or as starting components for other types of materials, envisaging applications that aim to prevent contamination of water sources and/or their treatment, mainly when CEC are involved. The revision of scientific work presented here was also framed by the research topics that our own group has developed on this subject. The several examples presented demonstrate the enormous potential of colloidal nanomaterials in applications aimed at water quality improvement. However, they are in distinct stages of potential application and, in certain cases, mainly regarded as interesting systems for acquiring new scientific knowledge. This is due not only to the nature of nanomaterials and their differentiated complexity, but also to the requirements that apply to each of the technologies in which nanomaterials are used. As an example, it should be noted that the scale at which nanomaterials are used differs between an application as nanosorbent in a water treatment plant and an analytical kit for laboratorial monitoring a target contaminant.Although research on nanomaterials as addressed in this work is in different stages of maturation, it is possible to highlight some challenges and possible lines of investigation aimed at their solution. Nanosorbents have clear advantages over conventional materials, however aspects related to selectivity for CEC molecules and subsequent regeneration, if necessary, remain important challenges. The application of colloidal materials in photocatalysis has focused mainly on the use of the UV region of the electromagnetic spectrum, thus complementary and innovative strategies are needed aimed at extending the photocatalytic activity to the visible region. The study of chemical speciation of solutions submitted to photocatalytic treatment is of increasing importance, because total mineralization of pollutant molecules has not been always demonstrated. The use of nanomaterials as adsorbents and in photocatalysis, in a real context of water cleaning technology, must be preceded by environmental impact studies. The strategy of associating magnetic phases either to adsorbents or photocatalysts, is a possible solution to ensure the development of safe and more sustainable processes based on magnetic separation technologies. Another alternative strategy could be the immobilisation of nanomaterials in membranes using new processing technologies that take advantage of the properties of colloidal nanoparticles. With regard to the development of SERS sensors for CEC detection, in addition to the detection limit and selectivity for target analytes, there is a need for quantitative analytical data for specific scenarios. This aspect requires even more fundamental research that integrates, in addition to the aspects of synthesis of plasmonic nanomaterials, the mechanisms associated to their interaction with the analyte molecules, namely by taking into account the nanoscale characterization of the substrate, which impacts on the uniformity and reproducibility of the SERS signal.In addition, there is a need for pilot projects aimed at water improvement applications for the most promising colloidal nanomaterials, in a realistic context, by selecting specific situations in which these materials can be part of the solution, in whole or in part. This type of approach also requires further ecotoxicity studies, also under realistic conditions, that allow the safe use of nanomaterials for the specific application for which they are intended and, in the required quantities. Finally, it should be noted that the unique properties observed in some of the nanomaterials reviewed here, can also be explored in other research contexts and innovative applications.


BackgroundNasogastric tube insertion is a routinely performed technique in anesthetized and intubated patients. However, failure leads to repeated insertion attempts causing injury, which makes the situation cumbersome. Therefore, we introduce a simple technique of deflating endotracheal tube cuff for nasogastric tube insertion in such patients.MethodsEighty adult patients scheduled for elective abdominal surgeries were randomly allocated into two groups, Group A  and Group B  along with lateral pressure application at cricoid in both groups. The number of attempts required, overall success rate, the time taken, rescue maneuvers, and incidence of complications were compared between the groups.ResultsIn Group B, the total success rate for nasogastric tube placement was significantly higher than in Group A . Group B had a 55% first attempt success rate, but only 25% of patients in Group A had a first attempt success rate (p=0.014). The overall time for successful nasogastric tube placement was longer in Group A than in Group B . The differences in nasogastric tube kinking and coiling between Group B and Group A were not statistically significant . Mucosal bleeding was substantially higher in Group A than in Group B .ConclusionsThis study showed that endotracheal tube deflation significantly increased the first-attempt success rate, overall successful placement of the nasogastric tube in intubated patients, and incidence of complications like mucosal bleeding decreased. The insertion of a nasogastric tube is an important procedure in abdominal, thoracic, and laparoscopic surgeries. It is a routinely performed procedure in intubated patients in the operating room. However, nasogastric tube insertion in an anesthetized and intubated patient is a tough job, with a high failure rate of around 50% on the first attempt using conventional technique . In the Many techniques have been suggested in the past to overcome the difficulty of conventional techniques such as head flexion ,2, laterStudy designAfter receiving approval from the Biomedical Research Ethics Committee, Pandit Bhagwat Dayal Sharma Post Graduate Institute of Medical Sciences, Rohtak, India , the current prospective, randomized interventional study was carried out in the Department of Anesthesiology and Critical Care. This study is registered with the clinical trials registry of India (CTRI/2022/01/039285).Study populationThis study comprised 80 patients between the ages of 18 and 60 who were scheduled for elective abdominal procedures and had an American Society of Anaesthesiologists (ASA) physical status I or II, as well as Mallampatti Grade (MPG) I or II, requiring nasogastric tube insertion. Patients with a history of bleeding disorders, history of head and neck deformities, the base of skull fracture, anticipated difficult airway, radiotherapy to head and neck area, history of nasal deformity or obstruction, and patients with esophageal varices were excluded from the study.InterventionPatients were randomly assigned to two groups using a sealed opaque envelope approach after receiving informed and written consent. Group A followed the nasogastric tube insertion without endotracheal cuff deflation, and Group B adopted the nasogastric tube insertion with endotracheal tube cuff deflation. In both groups, lateral pressure was applied at the level of the cricoid ipsilateral to the nostril in which the nasogastric tube was inserted. This was done to prevent the impaction of the nasogastric tube in the piriform sinus as it obliterates the potential space.Preanesthetic evaluation was done a day prior to surgery. A nasal patency test was done preoperatively to find the more patent nostril and rule out nasal deformities like a spur or deviated nasal septum. Patients were induced using an injection of fentanyl, propofol, and a non-depolarising neuromuscular blocking agent. All the patients underwent orotracheal intubation to secure the airway with an appropriate size tube. Sevoflurane and oxygen in nitrous oxide were used to maintain anesthesia (50:50).After tracheal intubation, xylometazoline nasal drops were instilled into both nostrils. An anesthetist with a minimum of five years of experience did nasogastric tube insertion. In both groups, a sterile, well-lubricated 14 French ROMSONS  nasogastric tube was inserted via a more patent nostril until it reached a length of 10 cm. Then, lateral pressure was applied to the neck around the cricoid area on the side similar to the nostril in which a nasogastric tube was inserted in both groups. In Group A, nasogastric tube placement was done through the more patent nostril without deflating the endotracheal tube's cuff. In Group B, nasogastric tube placement was done through the more patent nostril after deflating the endotracheal tube's cuff. The endotracheal tube's cuff was inflated after the correct placement of the nasogastric tube was confirmed by auscultation with a stethoscope in the epigastrium. If the first attempt failed, the nasogastric tube was withdrawn, cleaned, re-lubricated, and inserted into the same nostril using the same technique previously used. The number of attempts was noted along with the overall success rate. It was considered a failed placement if more than two attempts were needed.If more than two failed attempts occurred, the nasogastric tube was guided into the esophagus using Magill forceps under laryngoscope guidance as a rescue maneuver. The time was calculated from when the nasogastric tube was inserted through the nostril until it was successfully placed. Complications like kinking, coiling, or bleeding was noted in both groups.The primary goal of this study was to see if there was a difference in the overall success rate of both approaches for inserting a nasogastric tube and the first-attempt success rate. The secondary goal was to compare the time required for the successful placement of the nasogastric tube, the incidence of mucosal bleeding, kinking, coiling, and any other complications.Sample size calculationA previous study by Mahajan et al. suggested a 50% incidence success rate using the conventional technique of nasogastric tube insertion . TherefoStatistical analysisThe data gathered was entered into a Microsoft Excel spreadsheet . The Statistical Package for the Social Sciences (SPSS) version 20.0  was used to conduct all analyses. An unpaired t-test was used to examine age, body mass index (BMI), and nasogastric tube insertion time. In addition, ASA status, MPG grading, sex, number of attempts, successful insertion, and complications after nasogastric tube insertion were compared using the chi-square test. The statistical significance level was chosen at p=0.05.Our research comprised a total of 80 patients. The study's flow diagram is presented in Figure . There wIn Group A, the first-attempt nasogastric tube insertion success rate was 25%, whereas in Group B it was 55%. A second attempt was required in 30% of Group A cases and 25% of Group B cases (p=0.014). The overall success rate in Group B was significantly higher, at 80% vs 55% in Group A (p =0.016). The nasogastric tube was ultimately placed in the remaining cases in both groups using the rescue technique. The rescue technique was used in 18 (45%) patients in Group A and eight (20%) patients in Group B (p=0.0169). In Group A, the mean time for successful insertion was 65.4± 28.72 seconds compared to 43.5 ± 25.37 seconds in Group B (p=0.0005)  had bleeding episodes, whereas in Group B only four patients (10%) had bleeding episodes. Group A had considerably more bleeding (p=0.025). In Group A, kinking of the nasogastric tube occurred in nine patients (22.5%), whereas in Group B eight patients (20%) encountered kinking. Coiling of the nasogastric tube occurred in 11 (27.5%) cases in Group A vs 4 (10%)cases in Group B. Incidence of complications like kinking and coiling was not statistically significant between the groups   significantly improved the first-attempt success rate of nasogastric tube passage compared to the group where the endotracheal tube cuff was kept inflated (Group A). In Group A, 25% of patients had first-attempt successful placement of nasogastric tube as compared to 55% in Group B. Overall success rate was also significantly high in Group B (80%) when compared to Group A (55%) with a p-value of 0.01  found that lateral pressure and neck flexion improved the success rate of nasogastric tube placement in intubated patients undergoing laparoscopic hysterectomies . Illias 017 foundThe time required for successful nasogastric tube insertion in Group B was significantly less than in Group A (p=0.0005) (as seen above in Table In the present study, the incidence of complications like kinking and coiling was not statistically significant between the two groups (as seen above in Table This study had certain limitations. First, the performing anesthetist was not blinded to the type of intervention used. Second, this study was limited to ASA status 1 or 2, with Mallampatti Grade 1 or 2. Third, radiological confirmation remains the gold standard, but we used the auscultation method to confirm correct placement. Fourth, deflating the endotracheal tube is of concern in patients with inadequate fasting and patients who are prone to aspiration like in pregnancy and severe obesity, etc. So this technique is of limited use in such scenarios. Fifth, this research did not consider various other factors such as the size of the tongue, thyromental distance, and the height of the head ring/pillow. Further studies designed to overcome the constraints mentioned above are recommended. It would remain future scope to conduct trials in patients with limited neck mobility and difficult airway.We believe the endotracheal tube cuff deflation technique is a simple and feasible technique for nasogastric tube placement in anesthetized patients. It does not require any skill or instruments for nasogastric tube placement. This is the first study to evaluate the role of endotracheal tube cuff deflation in facilitating nasogastric tube placement, and we found favorable outcomes.The endotracheal tube cuff deflation-assisted technique improved the chances of successful nasogastric tube placement. It also helped to decrease complications like bleeding by avoiding multiple attempts. Therefore, we recommend using this innovative approach for nasogastric tube insertion in anesthetized and intubated patients.


The adverse effects of cardiopulmonary bypass during open cardiac surgery, including hemodilution, seem to be inevitable, especially for patients who generally have a relatively lower BMI with relatively small blood volumes. This study reports the modification and use of a cardiopulmonary bypass (CPB) system to reduce priming volume and hemodilution.This is a retrospective study of 462 adult patients who underwent cardiac valve replacement surgery from January 2019 to September 2021 at the General Hospital of Western Theater Command. The modified group consisted of 212 patients undergoing modified CPB. The control group included 250 patients receiving conventional CPB. Evaluated indices included fluid intake and output volumes during CPB, intraoperative indices related to CPB operation, usage of blood products during the peri-CPB period, and postoperative outcomes.P < 0.05) and colloid priming volumes , and ultrafiltration solution volume . Furthermore, the modified group had a significantly lower rate of defibrillation . The intraoperative urine volume  and intraoperative hematocrit (Hct)  of the modified CPB group were also higher than in the control group. The modified group required a lower infusion volume of packed red blood cells  and lower infusion rates of packed red blood cells  and fresh frozen plasma . In addition, the modified group showed significantly improved indices related to postoperative recovery.The modified group displayed a significant reduction in the crystalloid (200 mL vs. 600 mL, The modified CPB system effectively conserves blood and shows noteworthy potential for application in cardiac valve replacement surgery.The online version contains supplementary material available at 10.1186/s13019-023-02175-8. Cardiopulmonary bypass (CPB) remains an important tool for circulatory support in cardiac valve replacement surgery, but its adverse effects on blood seem to be inevitable, especially for patients with a lower BMI and relatively small blood volumes. Extreme hemodilution caused by large priming volumes decrease colloid osmotic pressure, resulting in the transfer of intravascular fluid to the tissue gap. The accumulation of extravascular fluid induces interstitial edema and corresponding organ dysfunction , 2. In aTraditional blood conservation efforts include a variety of techniques, such as preoperative anemia management , antifibNevertheless, MiECC has not been extensively applied worldwide, owing to some unresolved issues, such as its relatively complex operation and high cost. Moreover, abandoning the venous reservoir is a controversial measure as while it decreases the priming volume, it further increases the difficulty of emboli management during CPB. For cardiac surgeries associated with higher bleeding rates, such as cardiac valve replacement surgery, the absence of a venous reservoir also leads to excessive intraoperative blood loss and increases the required volume of blood transfusion. More importantly, the above drawbacks may have greater impact on patients with a relatively lower BMI with relatively small blood volumes. Therefore, modifying conventional CPB (CCPB) may offer more practical value. Some pioneering studies have reported modifications on CCPB, such as the minimization of CPB circuits and the integration of arterial line filters, which have further improvements in performance specifications. As a result, priming volume was decreased by approximately 500 mL, which led to a red blood cell transfusion rate as low as 13.6% .Inspired by these previous studies, we introduce a novel strategy for modifying the CCPB system to optimize blood conservation in adult cardiac valve replacement surgery through median sternotomy. We hypothesized that this modified CPB may reduce the priming volume of CPB devices, intraoperative hemodilution, and postoperative transfusions, all of which are favorable for intraoperative blood conservation and postoperative outcomes in patients undergoing cardiac valve replacement.Data were retrospectively collected from 462 adult patients who underwent cardiac valve replacement surgery from January 2019 to September 2021 at the General Hospital of Western Theater Command. The inclusion criteria were as follows: (1) age > 18 years; (2) patients with valvular heart disease diagnosed by cardiac color Doppler ultrasound; and (3) patients undergoing cardiac valve replacement surgery through median sternotomy in our department. The exclusion criteria were as follows: (1) patients who underwent emergency or redo cardiac surgery; (2) preoperative hematocrit (Hct) < 30; (3) patients requiring preoperative blood transfusion; and (4) patients requiring concurrent coronary artery bypass grafting or vascular surgery. The patients from January 2019 to June 2020 received conventional CPB during the surgery, while patients from July 2020 to September 2021 received modified CPB. After exclusion, 212 patients were included in the modified group. The control group consisted of 250 patients who met the same exclusion criteria. The patients in the control group were matched in terms of sex, age, weight, height, body mass index (BMI), preoperative Hct, diagnosis, surgical procedures, and concomitant surgeries distribution for comparisons with the modified group. This study was approved by the Institutional Ethical Review Board of the General Hospital of Western Theater Command (2020ky013).Information regarding the reagents and materials used for the CPB system is provided in the Additional file The CCPB system comprised roller pumps, an oxygenator, an arterial line filter, cardioplegia, and its perfusion circuit. The main pump tubing, a tubing connecting the venous reservoir with an oxygenator and passing through the main pump, was polyvinyl chloride (PVC)/silicone composite tubing with a total length of 210 cm, in which the diameter of the PVC and silicone sections were 10 mm and 12 mm, respectively. The arterial and venous ports of the oxygenator were connected with PVC tubing. The venous tubing was 12 mm in diameter and 135 cm in length, and the arterial tubing was 10 mm in diameter and 120 cm in length. The diameter of the tubing from the outlet end of the oxygenator artery to the arterial line filter was 10 mm and the length was 75 cm. Thus, the total length of the CCPB circuit was 540 cm. The CCPB system adopts gravity drainage mode, and the venous reservoir is 10 cm away from the ground. Histidine-tryptophan-ketoglutarate (HTK) solution was applied in the CCPB for cardioplegia. The single perfusion dose of cold HTK solution (4–8 °C) was 30 mL/kg. A suction tube was placed in the coronary sinus to remove the discharged fluid and prevent the HTK solution from entering the blood circulation. The perfusion pressure was maintained at 100 mmHg before cardiac arrest. After cardiac arrest, the perfusion pressure was reduced to 50 mmHg to ensure complete perfusion of the remaining solution. The total perfusion time was kept between 5 and 7 min. The perfusion circuit of cardioplegia included a cold filling device, a rolling pump, and a perfusion needle, which were connected by PVC/silicone tubing with a total length of 300 cm. In order to recover intraoperative bleeding as much as possible, the CCPB system was also equipped with left atrial suction and an intracardiac suction.As shown in Fig. Shortening the CPB tubing lengths: To reduce priming volume, the modified CPB system featured a shortened main pump tubing of 80 cm, arterial tubing of 100 cm, and venous tubing of 110 cm. The total length of the system tubing was 290 cm, 250 cm shorter than that of the CCPB system.Incorporating an arterial line filter into the oxygenator: The modified CPB system used an oxygenator that integrated an arterial line filter, and the position of the oxygenator was raised to 80 cm above the ground to minimize the length of the tubing between the oxygenator and the main pump Fig. B.Use of a VAVD device: To ensure adequate venous drainage, the modified CPB system featured a VAVD device added to the venous reservoir and continuously monitored venous end pressure.the liquid storage bag was connected to the tee connection of the side branch circulation of the oxygenator. After this, the rolling pump was started to discharge the priming solutions in the venous reservoir into the liquid storage bag, and the pump was stopped when the liquid level of the venous reservoir was 100 mL.After successful aortic cannulation, the arterial clamp was opened, slowly making the patient's arterial blood retrograde, replacing the priming solutions in the arterial tube, and discharging the priming solutions in the arterial tube into the liquid storage bag from the tee connection of the collateral circulation of the oxygenator.After successful venous cannulation, the venous clamping forceps and VAVD device were opened, using negative pressure to discharge the priming solutions in the venous tubing into the venous reservoir, the venous tubing was clamped immediately after the blood filled the venous tubing, the VAVD device closed, and the rolling pump restarted to discharge the priming solutions in the venous reservoir into the liquid storage bag.Finally, the venous tubing and VAVD device were opened, a small portion of venous blood was drained into the venous reservoir, the rolling pump began to make the blood in the venous reservoir enter the main pump tubing, and the priming solutions discharged into the liquid storage bag to complete the entire operation.Adopting the retrograde autologous priming method: To further reduce priming volume, a retrograde autologous priming step was added during CPB preparation. Deoxyepinephrine (5 µg) was first applied before the operation to maintain the patient's blood pressure above 100 mmHg. If systolic pressure fell below 50 mmHg, the retrograde autologous priming operation was aborted and the patients were excluded from the modified group. In this study, all 212 patients in modified group tolerated the retrograde autologous priming.2 flush procedure to the CPB circuit before surgery: During the CPB system assembly, the tubing was flushed with CO2 for > 10 min to eliminate air in the device.Addition of the COCompared with the CCPB system, our modified CPB system featured the following improvements:All patients were treated with the same anesthetic induction and maintenance procedure, surgical approach, and CPB management strategy. The surgical details are described in the Additional file Four fluid intake and output volumes were measured during CPB: crystalloid priming volume (mL), colloid priming volume (mL), ultrafiltration solution volume (mL), and intraoperative urine volume (mL). The amount of fluid given prior to bypass was approximately 100 mL in both groups, the majority of which was the liquid volume of the narcotic drugs used.The intraoperative indices related to CPB were measured, including cardiac surgery time (min), CPB operation time (min), cross-clamp time (min), net volume of infused cardioplegia (mL), rate of defibrillation (%), pre-CPB lactic acid (Lac) (mmol/L), 1-h intraoperative Lac (mmol/L), post-CPB Lac (mmol/L), pre-CPB Hct (%), 1-h intraoperative Hct (%), post-CPB Hct (%).9/L.Blood volume return from the CPB device (mL), the infusion volume of packed red blood cells (mL), and the infusion rate of different blood products (%) were measured. The peri-CPB period refers to the period from the beginning of CPB to hospital discharge, in which the amount of transfusion immediately after bypass or intraoperatively is also included. Blood volume return from the CPB device refers to the blood volume that remained in the extracorporeal circulation after bypass. The packed red blood cells were infused when the patient's Hct was less than 20% during surgery or less than 24% while in the intensive care unit (ICU). During the hemostasis process of surgery, Fresh frozen plasma (FFP) was infused when the anesthesiologist determined that the patient has hypotension caused by insufficient volume. In ICU, FFP was infused when the R and K values of thromboelastogram significantly increased and the MA and Mε values of thromboelastogram significantly decreased. In addition, the drainage volume of patients was considered to maintain effective circulation volume. Platelets were infused when the patient had obvious bleeding indication or the number of platelets was less than 30 × 10The 12-h postoperative Hct (%), 12-h postoperative Lac (mmol/L), incidence of complications, ICU results, and post-operative total length of stay were also collected.t-test was used for the comparison between groups. Non-normally distributed continuous variables were expressed as median (interquartile range), and a nonparametric test was used for the comparison between groups. Categorical variables were expressed as frequencies and percentages. The P value below 0.05 was recognized as statistically significant for each test.The Kolmogorov–Smirnov method was used to test the normality of the continuous variables. Normally distributed continuous variables were expressed as mean and standard deviation, and a P = 0.497). Most patients required valve replacement for rheumatic heart disease, but other indications included congenital valvular disease and degenerative valvular disease. All patients underwent aortic valve replacement, mitral valve replacement, or double valve replacement. Some patients underwent concomitant surgeries, such as concurrent tricuspid valve repair or left atrial plication. The two groups did not differ in their diagnoses, surgical procedures, and concomitant surgeries     . The modified group had a significantly higher net volume of infused cardioplegia  compared with the control group. The modified group had a significantly lower rate of defibrillation  compared with the control group, indicating better intraoperative myocardial protection. To evaluate whether intraoperative circulatory perfusion was appropriate, some key indices of arterial blood gas analysis were investigated. No significant differences in pre-CPB Lac, 1-h intraoperative Lac, post-CPB Lac, or pre-CPB Hct levels were observed between the two groups, whereas the modified group had significantly a higher 1-h intraoperative Hct  and post-CPB Hct .As shown in Table P < 0.05). With respect to the use of specific blood products, packed red blood cells were administered to 38 patients (17.9%) in the modified group versus 63 patients (25.2%) in the control group (P < 0.05). Only three patients (1.41%) received plasma in the modified group, compared with 13 control group patients (5.2%) (P < 0.05). There were no significant differences in platelet infusion rates between the two groups (P = 0.835).As shown in Table P < 0.05). No significant differences in the 12-h postoperative Lac were observed between the two groups. Some patients in the two groups developed cerebrovascular events or reoperation for postoperative bleeding, but there were no significant differences in their incidence . Additional procedures, such as continuous renal replacement therapy (CRRT), intra-aortic balloon pump (IABP) treatment, or extracorporeal membrane oxygenation (ECMO) treatment, were not required in the modified group. In the control group, two patients (0.80%) received IABP treatment, and one patient (0.40%) received ECMO treatment. There were no significant differences between the groups regarding CRRT (P = 1.000), IABP treatment (P = 1.000), and ECMO treatment (P = 1.000). With respect to ICU outcomes, the modified group required less ventilation time than the control group , but no significant differences in time spent in ICU were observed between the two groups . In addition, no significant differences in post-operative total length of stay were observed between the two groups .Table In this study, we reported the modification and use of a CPB system to reduce the priming volume and conserve blood in adult cardiac valve replacement surgeries. Briefly, the tubing length was shortened by 250 cm in our modified CPB, and approximately 850 mL less priming solution was required compared with the CCPB system. This decrease was more than the value reported in a similar modified-CPB study, which decreased the priming volume by approximately 500 mL , 16. TheAfter decreasing the priming volume, the modified CPB system exhibited an attractive blood-conservation effect. Higher 1-h intraoperative Hct and 12-h postoperative Hct were observed in patients in the modified group. Additionally, the infusion rates of packed red blood cells and fresh frozen plasma decreased by 7.3% and 3.8%, respectively. These results indicate that the lower priming volume used in the modified CPB system reduced hemodilution. Successful attempts to minimize hemodilution and avoid blood transfusion by decreasing the priming volume have also been reported previously. A univariate analysis revealed that CPB circuit priming volume is a potentially modifiable risk factor for the need for blood transfusion in patients undergoing cardiac surgery with CPB . A similSome intraoperative and postoperative outcome indices were also observed in this study. The differences between the groups were statistically non-significant regarding cerebrovascular events, treatment with IABP and ECMO, CRRT, and ICU stay time. A lower rate of defibrillation and shorter ventilation time were observed in patients in the modified group. Nevertheless, the shorter ventilation time seems to be due to the surgical management. Nowadays, patients with a problem-free course can be extubated on-table, using the fast-track concept. Similar ideas apply here for ICU stays.2 flush procedure contribute to a reduction of gaseous microemboli in CPB devices and corresponding improved neuropsychological outcomes [2 flush procedure before surgery in our modified CPB system. However, the differences between the two groups were not statistically significant regarding cerebrovascular events in our study. Potential biases of this result were the limited number of patients experiencing cerebrovascular events, the influence of CO2 flushing of the cardiac chambers during surgery, and the non-uniform administration technique for CO2 flushing. Therefore, the practical value of the CO2 flush is yet to be fully investigated. Our future work will aim to further explore its efficacy by expanding the number of patients and observation indicators regarding the remaining air in the CPB circuit, gaseous microemboli, and neuropsychological outcomes. Such studies may reveal other clinical improvements above blood conservation, such as cerebral protection. In addition, the lower incidence of defibrillations may suggest better myocardial protection, but the mechanism cannot be explained with a clamped aorta. We speculated that the lower incidence of defibrillations may be related to the improved blood conservation in the modified CPB group. Blood with a higher Hct may favor oxygen delivery to the myocardium in the early stage of CPB. However, we were unable to determine the detailed mechanisms in our study as there is no way to evaluate the impact of Hct on myocardial oxygen consumption during CPB. In addition, the lower incidence of defibrillations alone cannot conclude improved myocardial protection. Potential bias, such as the de-airing of the heart during surgery, should be excluded. More parameters representing myocardial protection should be observed in the future, including cardiac index or myocardial injury markers.Compared with previous work, the fabricated CPB system in this study included several improvements, but inevitably had limitations. Previous studies have found that adding the COoutcomes , 25, 26.In summary, a modified CPB system was fabricated and applied to adult cardiac valve replacement surgery. With the introduction of five optimization measures, the modified CPB system saved approximately 850 mL of priming solution and presented an attractive blood conservation effect compared with CCPB. Both the intraoperative and postoperative results revealed that the modified group exhibited a higher Hct, indicating less hemodilution. It also resulted in a reduction for the need of specific blood products, and infusion rates of packed red blood cells and fresh frozen plasma decreased by 7.3% and 3.8%, respectively. With the aid of the modified CPB system, a lower rate of defibrillation and shorter ventilation time were achieved. As a result, the clinical effectiveness of this CPB system, including myocardial protection, may be envisioned. These findings demonstrate that the modified CPB system described herein greatly conserves blood, with a noteworthy potential for application in cardiac surgery.Additional file 1: Supplementary material includes the reagents and materials used for the CPB system, the anesthetic procedure, surgical techniques, and CPB management strategies.


Raphanus sativus. In contrast, these modifications were either absent or detected only at trace quantities, across all yeasts and insect genomes studied. Further, we reveal interesting biological cases. For instance, in Allium cepa, Helianthus annuus, or Andropogon gerardi, more than 35% of cytosines were methylated. Additionally, next to the mammlian CNS, 5hmdC was also detected in plants like Lepidium sativum and was found on 8% of cytosines in the Garra barreimiae brain samples. Thus, identifying unexpected levels of DNA modifications in several wild species, our resource underscores the need to address biological diversity for studying DNA modifications.Interpreting the function and metabolism of enzymatic DNA modifications requires both position-specific and global quantities. Sequencing-based techniques that deliver the former have become broadly accessible, but analytical methods for the global quantification of DNA modifications have thus far been applied mostly to individual problems. We established a mass spectrometric method for the sensitive and accurate quantification of multiple enzymatic DNA modifications. Then, we isolated DNA from 124 archean, bacterial, fungal, plant, and mammalian species, and several tissues and created a resource of global DNA modification quantities. Our dataset provides insights into the general nature of enzymatic DNA modifications, reveals unique biological cases, and provides complementary quantitative information to normalize and assess the accuracy of sequencing-based detection of DNA modifications. We report that only three of the studied DNA modifications, methylcytosine (5mdC), methyladenine (N6mdA) and hydroxymethylcytosine (5hmdC), were detected above a picomolar detection limit across species, and dominated in higher eukaryotes (5mdC), in bacteria (N6mdA), or the vertebrate central nervous systems (5hmdC). All three modifications were detected simultaneously in only one of the tested species,  Arabidopsis thaliana has orders of magnitude higher levels 5mdC compared to the dominant insect model Drosophila melanogaster, while the dominant yeast model organism Saccharomyces cerevisiae lacks this modification altogether , represent the rule or the exception in their respective phylogenetic group without a broader multi-species dataset for comparison.Until recently, studying DNA modifications was technically challenging, information concerning their content and function was scarce for species other than model organisms, several crops, and humans. Moreover, it was rather difficult to translate the knowledge derived from those intensively studied species into a broader biological context. For instance, it is hard to judge from the current literature if the low amount of DNA modifications in laboratory yeast and In addition to the position-specific information provided by sequencing technologies , global In order to quantify the global levels of multiple enzymatic DNA modifications in a single analysis, we expanded a previous method based on liquid chromatography-multiple reaction monitoring (LC-MRM) and designed for the quantification of 5mdC . This meMus musculus), African clawed frog (Xenopus laevis), but also for some less studied species, the opossum (Monodelphis domestica), the Alpine marmot (Marmota marmota), and the Oman garra (Garra barreimiae), we obtained DNA from multiple tissues and/or cell lines in order to quantify tissue differences in the absolute DNA modification content. For plants, we focused on seedlings that were germinated in the lab  which is chemically mild to DNA, and avoided strategies that involve the use of oxidants and reactive chemicals. However, for plant species, due to their biochemical composition, we were forced to use phenol–chloroform extraction to obtain sufficient quantities of DNA. In such cases, reagents like β-mercaptoethanol (2-sulfanylethan-1-ol) were included to keep DNA damage to a minimum during the extraction. The obtained DNAs were from 124 different species, including 85 bacterial species, 6 yeast species, 2 archeal species, 19 plant species, and 18 tissue and cell-culture samples from multiple animal species, including human and mouse. The collection included both the typical model organisms, and specifically for bacteria, vertebrates, and plants we included a significant number of species that have been barely characterized at the molecular level so far . Further the lab . The see the lab .Trichoplusia ni, Spodoptera frugiperda, and D. melanogaster  principally contained methylated cytosine as the preferred base modification .The presence of other DNA modifications in  results . We asse ~0.04%, . In a revolution . While tDiplotaxis tenuifolia had low amounts of N6mdA , which shared a similar level of the cytosine modification but differed in their levels of adenosine modification. While we detected N6mdA in H. salinarum, no adenosine modification was observed for S. acidocaldarius  of the cytosine residues. Some species, including the model organisms rvations . In planrvations . Extremethylated . As planthylated . Given tX. laevis, M. musculus), and three non-model organisms . From M. musculus we further examined tissues from multiple inbred laboratory lines: BALB/c, FVB/N, Hsd/Ola/MF1, B6SJL/CD451/CD452, BALB/cAnN, 129S8, and F1/CBAxB6. In parallel, we analyzed multiple human cell lines  and X. laevis, respectively, were different to the aforementioned species. In opossum, we detected much lower levels (2%) of 5mdC in all tissues examined. Conversely, in X. laevis, all tissues had much higher concentrations (about 9.4%). Higher values in X. laevis could be attributed to the tetraploid genome of this species compared to its relative X. tropicalis, which is diploid (Phaseolus vulgaris and obtained consistently high (16.7%) 5mdC concentrations in all measured tissues , and amphibian X. laevis (2%) still had high levels of 5hmdC specifically in brain tissue relative to other tissues in those organisms  in which we confirmed low concentrations of 5hmdC. Furthermore, we did not detect 5hmdC in any of the bacterial or fungal genomes analyzed. Our results support the fact that the modification of 5hmdC is more widespread in biological systems than previously assumed, but quantities above picomolar-levels being not detected in any bacteria, yeasts and many tissues from higher organisms implies 5hmdC is not universal or specific to any part of the phylogenetic tree.Apart from vertebrates, 5hmdC was also observed in a sativa . Our datMobiluncus curtisii (~1.4%) and Moorell thermoacetica (~1.1%). In total, the prokaryotic genomes hence contained higher amounts of DNA modifications compared to lower eukaryotes such as yeasts and insects, but lower amounts of DNA modifications compared to higher eukaryotes—plants and vertebrates in particular. Typical bacterial species contain only one type of modification—mostly N6mdA (Campylobacter contain trace quantities of 5mdC (<0.1%) next to the dominating N6mdA modification , has only 0.1% of its adenines methylated. Interestingly, we observed that median values for 5mdC dominate over N6mdA in those bacteria that colonize or enter mutualistic relationships with higher eukaryote species that carry 5mdC as their main modification  of cytosine residues were methylated while containing only <0.3% N6mdA, and Faecalicoccus pleomorphus and Bifidobacterium adolescentis, with >1.5% of 5mdC without any detectable levels of N6mdA modification. Indeed, others made a similar observation in single-cell fungi. While the environmental yeasts studied herein and previously lacked any modifications  at 37°C for 45 min, and DNA purification was performed using QIAGEN Genomic tip-20/G according to the manufacturer’s instructions. Purified DNA was precipitated with isopropanol, washed with 70% ethanol, and resuspended in 10 mM Tris-HCl, pH 8.0. Quantification was done using a dsDNA BR Assay Kit (Qubit). The DNA sample was then digested into corresponding nucleosides using DNA Degradase Plus . 1 µg of DNA was treated with 5 U of DNA degradase at 37°C for 2 hr in a final volume of 25 μl and the reaction was inactivated by incubating the samples for 20 min at 70°C as described by the manufacturer. Calibration standards were prepared in 1:4:4:2:2:2:2:4:4:4:4:4:4:4:10 serial dilutions from a standard stock that was prepared as per The samples were diluted 1:1 with MeOH 10% (v/v) containing 0.2% formic acid, and 10 µl corresponding to 200 ng of gDNA were injected onto a liquid chromatography system equipped with reverse phase Acquity UPLC HSS T3 column, 100 Å, 1.8 µm, 2.1 mm × 150 mm (Waters), column temperature 25°C and flow rate of 0.2 ml/min. Mobile phase A: 0.1% formic acid +10 mM ammonium formate in water, mobile phase B: 0.1% formic acid +10 mM ammonium formate in methanol. Gradient for elution was started from 5% mobile phase B to 35% B over 11.5 min followed by sharply increasing to 80% over the next 1.5 min. The gradient was held at 80% B for 2 min, lowered to the starting gradient over 1 min, and equilibrated for 6.5 min. Total length was 22.5 min.The eluent was directed to an electrospray ion source connected to a triple quadrupole mass spectrometer (Agilent 6470 QQQ) equipped with an Agilent Jet stream source, operating in positive mode. The ESI source settings were: gas temperature: 300°C; gas flow: 6.4 l/min; nebulizer: 50 psi; sheath gas heater: 350°C; sheath gas flow: 7 l/min; capillary: 2000 V. The transitions monitored for MRM experiments are listed in For neutral loss experiments, the samples were injected as per the same LC parameters used for the MRM experiment while the mass spectrometer was set to a scan type of neutral loss (M=116 Da) while scanning the quadrupoles from 230 to 250 Da. The scan time was 1000 with a step size of 0.05 amu and the values for Fragmentor, collision energy, and cell accelerator voltage were 73, 8, and 5, respectively. 4mdC was detected as the second peak in the neutral loss (Δ = 118) chromatogram corresponding to parent ion 242 Da.http://bacdive.dsmz.de, accessed April 14, 2020; Peak areas were extracted and integrated using MassHunter for QQQ to obtain the concentrations after applying the necessary limits of quantification. Subsequent processing for batch-to-batch variation and technical outlier removal were carried out using R or Python. A single reference mouse DNA sample was included in every measured batch to monitor batch-to-batch variation. Median-value-based normalization of the reference mouse samples was used to obtain the correction factor with which the corresponding batch was corrected. The results are depicted as percentage modification with respect to dG (for 5mdC and 5hmdC) and T (for N6mdA). The phylogenetic clustering was carried out using a newick file generated using NCBI Taxonomy (PhyloT) and the ggtree package . Feature DNA methylation is an important mechanism to control gene expression, yet methods for quantitation of global DNA methylation analyses are limited. This work provides a new sensitive method for the quantitation of global DNA methylation and they apply this to over 100 species of eukaryotes and prokaryotes, finding interesting differences across species. This is a useful tool and resource for those interested in DNA methylation and evolution. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Review Commons.]. This analysis generated values of DNA methylation that are not apparently consistent with previous datasets . It is difficult to understand the data of the new species analyzed and to make parallelism across those. The authors should consider using the total number of C or A recovered in the analysis of each sample and where possible compare and verify that this amount corresponds to the one annotated in the available genomes.For most of the species studied in our manuscript, there are no analytically determined absolute concentrations of DNA modifications found in the literature. Furthermore, is difficult to conclude from the local concentrations as determined by sequencing methods, on the real total amount of modified residues, specifically in species with complex genome structure, high ploidies, and lots of repetitive sequences. That is why one needs to measure these total concentrations with a direct and quantitative analytical method, as we have done it and our paper, and not extrapolate these values from indirect measures, like a genome sequence.– The method used to calculate DNA modification percentage, represented as normalized over G and T, does not take into account that the relative amount of G and T can differ in the respective genome/organism analyzed. When making comparisons directly across different species, the authors should consider to normalize and scale the total number of G or T present in each sample.We apologize for any confusion and if there was lack of clarity. In this method, we have of course not only measured the modified bases but also the unmodified ones . Therefore, the variation in the amount of G or T is reflected in the measurements and shouldn't influence the DNA modification percentage, and we can accurately represent the relative levels of modifications, irrespective of genome size.– The authors claim for Figure 3B "Our dataset shows that 5hmdC is detected in a broad range of vertebrate tissues except for spleen, but reaches significantly higher concentrations specifically in samples from the CNS". Even if the trend is clear the authors should consider using some statistical test to claim significant differences.Assuming a normal distribution, and have applied a simple T-test, which showed that the difference is highly significant.– In Figure 4B the authors state "At the phylum level the patterns were more prominent in Proteobacteria, containing more N6mdA than 5mdC, while a reverse trend of more 5mdC than N6mdA was observed for Bacteroidetes and Firmicutes". The reverse trend of Bacteroidetes and Firmicutes is not present as the median of the box plot is lower for 5mdC compare to N6mdA and the data have just bigger dispersion in that Phylum. As already mentioned, the authors should perform some statistical test to claim differences.Assuming a normal distribution, and have done analysis of variance, which showed that the difference is highly significant. The p values are given in the figure description.Minor comments:– For some of the plant samples, the authors use seeds as they represent an easier and cleaner source of genomic DNA. Is the analysis of DNA modifications in seeds comparable to "adult" fully developed tissues? Or is it different, as it happens when comparing gametes to adult tissues in animals?We would like to correct the reviewer that we use plant seedlings (not seeds). This unlike gametes have differentiated tissues. Our (admittedly limited) data of different plant tissues suggests that the total overall concentrations is quite similar between different tissues, a situation that is also observed in mammals. We discuss this in the revision.– Figure 4.B-C is not described in a linear way in the text, not reflecting the order in which the panels are positioned. Authors should consider reshuffling either the text or panels.We apologize for the lack of clarity. We have performed the necessary corrections.– In the text, there are many references to "Methods" section but this section is missing in the main text and that information are kind of reported altogether within the supplementary file. The authors should consider creating an appropriate Material and Method section in the main text.As the material section is quite large due to large sample size with their respective protocols, we had to move this section to the supplementary information file. But to aid the reader find the sections easier, we have now included the important aspects of the section in the main manuscript.– The common names and species names are used interchangeably; for readers to keep track, it would help to have a table listing the common names with the species names.We apologize for the inconsistency in the usage. To simplify, we have reverted to the usage of only scientific binomial names of all the species used in this study.– The authors should consider investigating deeper some of the interesting findings they observed in the group of bacteria hosted by higher eukaryotes that showed higher 5mdC compared to the free-living corresponding bacteria. This could be of interest not only at the epigenetic or molecular level but could also harbor a clinical relevance since some of them are able to induce human pathologies.As this manuscript was intended only as a resource manuscript and microbiology not being the specialty of our laboratory the request of the reviewer is out of scope- we hope however, that exactly such investigations are stimulated by our resource.