Background: Native breast milk composition displays significant inter- and intra-individual variation which persists after standard fortification with fixed doses and challenges target fortification. This study aims to analyze the macronutrient composition of different commercially available fortifiers and the effect of different fortification strategies on nutritional intake of preterm infants.Methods: In 103 preterm infants, native breast milk samples were collected from 24-h feeding batches  and fat, protein and carbohydrate contents were analyzed. Nutrient content was compared for breast milk that had undergone either (i) standard fortification, (ii) targeted fortification, (iii) selective batching according to breast milk composition, or (iv) partial lyophilization. For (i) eight commercially available standard fortifiers were tested. Targeted fortification (ii) involved the addition of single component modulars of either protein, fat or carbohydrates to standard fortified breast milk. Using a mathematical growth model, the combined effect of protein, fat and carbohydrate intake on growth was assessed. The best composition of standard fortifiers as the initial step for target fortification was explored assuming three clinical scenarios for milk analysis.Results: Macronutrient content was highly variable between native breast milk samples, and this variation was still present after standard fortification, however at elevated macronutrient levels. Standard fortification, breast milk batching, as well as partial lyophilization of human milk resulted in deficient and imbalanced enteral intakes in a significant proportion of infants. Target fortification reduced this variation in a, respectively, higher percentage of samples. The effect size was dependent on the number of measurements per week. The optimum composition of standard fortifiers was dependent on the clinical scenario (measurement frequency) for target fortification.Conclusions: To provide precise and accurate intakes of macronutrients, breast milk should be target fortified. Standard fortified breast milk can result in excess above recommended intakes of some macronutrients which limits the efficiency of target fortification. Standard fortifiers with improved composition are needed for target fortification. Between 43 and 97% of preterm infants experience postnatal growth restriction , 2. AdeqFortification of native breast milk is routinely used to meet clinical recommendations of enteral nutrient intake . VariousIn the current paper, we examine differences in macronutrient contents of native breast milk samples and the impact of standard and targeted fortification on this variation when using different standard fortifiers and analysis schedules. Further, in this study we aimed (1) to identify the best composition of a standard fortifier for three clinical scenarios for target fortification; (2) to analyze the effect of fortification on infant's growth based on established growth models; and (3) to study pre-fortification approaches to optimize the macronutrient content by batching and lyophilization.This modeling study is a secondary analysis of a dataset obtained during a randomized controlled clinical trial (RCT) which was conducted between November 2012 and July 2016 at the Division of Neonatology (Level-III NICU) at McMaster Children's Hospital  . The purn = 3,338) of 24-h feedings before breast milk was fortified. Batches were usually prepared from frozen milk of the own mother (MOM) and—depending on the level of available MOM—supplemented with pasteurized frozen human donor milk. Data from 103 preterm infants born at a mean gestational age of 27.0 ± 1.5 weeks  were available. Fortification (and subsequent collection of breast milk samples) began at day of life 21 ± 6 and continued over an average period of 33 days ± 10 days. Each study participant provided samples covering at least 14 consecutive days. The native breast milk samples were stored at −20°C until milk analysis.Samples were collected from pooled batches . Content of true protein and fat was determined by a validated near-infrared milk analyzer  , 30. LacLevels of fat, protein, carbohydrate, and lactose [g/100 mL] were measured in all native breast milk samples. The energy content  was calculated using energy equivalents which assume that protein and carbs yield 4 kcal/g and that fat yields 9 kcal/g of energy, respectively (Atwater factor) . AdditioTarget macronutrient intake for preterm infants <1,000 g of birth weight were defined according to the guidelines of the European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN): 4.5 g/kg/d protein, 6.6 g/kg/d fat, and 13.2 g/kg/d carbs . With anIn this study, seven commercially available multicomponent powdered standard fortifiers (fortifiers 1–7) with different compositions were used. Each of them provides 1.0–1.5 g of extra protein per 100 ml thereby comprising comparable energy intakes. Based on their non-protein composition, they can be divided into three groups: (i) low-fat/high-carbohydrate, (ii) medium-fat/medium-carbohydrate, and (iii) high-fat/low-carbohydrate content . For eacIn a second step, the optimum macronutrient composition for standard fortification was identified based on three different scenarios for milk analysis as described below (fortifiers 8–10). Composition was optimized for each macronutrient to minimize the deviation between fortified breast milk content and ESPGHAN recommended intake by minimizing the residual sum of squares. In a next step, the performance of fortifiers was evaluated for the three different measurement scenarios. Fortifier 8 represents a fortifier that was optimized for standard fortification only, i.e., no measurement of macronutrients in native breast milk. Fortifiers 9 and 10 were optimized for target fortification using either one breast milk analysis per week (occurring on Monday) or three breast milk analyses per week , respectively.Further, the macronutrient intake for human milk based liquid fortifiers (fortifiers 11a-d and 12) were also calculated. Fortifiers 11a to 11d represent the different preparation strengths (20–50 mL). Fortifiers 11 and 12 represent the same product, however, fortifier 12 uses different volumes as an off-label clinical approach.Target fortification was calculated using three steps . First, For the target fortification approach, we tested five different schedules for milk analysis and simulated the resulting macronutrient concentrations using fortifiers 1–12 .Macronutrient intakes achieved by using commercially available human milk based liquid fortifiers were also investigated. Different from powdered fortifiers, all liquid fortifiers inherently will replace a specific volume of native breast milk. For example, for a final feeding volume of 100 mL, 20 mL of fortifier product will be added to 80 mL breast milk. To achieve higher strengths of fortification the ratio of fortifier-to-breast milk needs to be increased . In this study, four different fortifier strengths were studied consisting of the following composition per 100 ml of ready-to-feed-batches: 20 mL (fortifier 11a), 30 mL (fortifier 11b), 40 mL (fortifier 11c), and 50 mL (fortifier 11d). All four different liquid fortifier products contain identical macronutrient concentrations (g/mL). The 10 mL- increase in the volume ratio of fortifier to breast milk leads to an incremental increase in protein by ~0.6 g, fat by ~0.9 g, and carbohydrates by ~1.0 g per 10 mL of fortifier.The following approaches were applied and referred to as fortifier 11 in the analyses: (1) Standard fortification with liquid fortifier: an average protein content of 1.2 g/100 mL in native breast milk was assumed. Fortifier 11b (30:70 v/v ratio) which increases the protein content by 1.8 g/100 mL was chosen as reference fortifier because it represents the fortifier which is most frequently used in clinical routine. (2) For target fortification: Breast milk analysis was performed and the extra protein content needed to reach 3 g/100 mL was calculated. Based on the analysis, the fortifier (11a to 11d) was chosen that was most appropriate to reach the protein target range of 2.8–3.4 g/100 mL. In the final step, fat and carbohydrate content—if below target—were adjusted by adding single component modular products to meet ESPGHAN recommended intake .Switching between the four different strengths of the HM based liquid fortifiers  will lead to stepwise increase of protein and macronutrients intake. To allow a more continuous increase fortifiers were dosed in an off-label approach to achieve ESPGHAN recommendations. This approach is categorized as fortifier 12.For this off-label approach, target fortification was performed by measuring breast milk macronutrient content and native breast milk batches were adjusted to final target protein concentration of 3 g/100 mL. For native breast milk with a protein content of ≥1.8 g/100 mL, liquid fortifier 11a was used as a 20: 80 ratio . For native breast milk with a protein content of ≥1.2 to 1.8, the liquid fortifier was 11a was used as the base component and fortifier 11b was added to reach a protein content of 3 g/100 mL. In this case, the ratio between the volume of fortifier 11a and 11b was 2:3 or, as expressed in volume referred to a ready to feed volume of 100 mL, 20 ml of fortifier 11a would be replaced by 15 ml of fortifier 11b, consequently, the volume of native milk needs to be reduced by this amount. For native breast milk with a protein content of ≥0.6 to <1.2 g/100 mL, fortifier 11b constituted the base fortifier and fortifier 11c was added in a ratio of 3:4. For native breast milk with a protein content of ≥0 to <0.6 g/100 mL, fortifier 11c formed the base component and fortifier 11d was added in a ratio of 4:5 .After target fortification of protein, the final step of adjusting fat to 4.4 g/100 mL and carbs to 8.8 g/100 mL was performed using modular products.Because of the natural intra- and interindividual variation of breast milk macronutrient composition standard fortification  will always result in over- or under-fortification of a significant number of breast milk samples. To investigate the magnitude of this inherent weakness feeds with inappropriate macronutrient composition shall be identified applying principles of nutritional physiology. Levels of all three macronutrients and energy as well as ratios of protein-to-energy as well as carbohydrate energy-to-non-protein energy were incorporated into an established prediction equation for growth velocity –35. FeedTwo nutritional physiological approaches were considered. The base equation was derived from Kashyap et al. . This eqwith energy intake as [kcal/kg/d], protein intake as [g/kg/d] and birth weight (BW) as (g). For our calculations a constant birth weight of 1,000 g was assumed.Growth rates are mainly determined by protein and energy intakes, but nutritional studies have shown that variations of the carbohydrate-to-non-protein energy ratio significantly modulate growth rates , 34. ThiTarget weight gain velocity was defined using (Equation 3) thereby assuming ESPGHAN recommended intakes for an infant with a body weight of 1,000 g , the target weight gain velocity was defined. Macronutrient composition of single feeds was considered as “inadequate” if the estimated weight gain velocity was >1 g/kg/d above or below the target weight.Human milk based liquid fortifiers were not included in this analysis due to insufficient reference data. Additionally, the fat phase from human milk might have different physiological effects when compared with fat in formula .n = 6 and n = 8 days (~1 week) to allow paired matching. The interval length was chosen assuming a minimum number of frozen milk samples during the major part of a NICU stay available to prepare feedings. Within each group BM samples were sorted by one macronutrient  or by energy content in ascending order. Assuming identical volumes , as a next step two or three samples were pooled using two different approaches:To investigate whether the natural variation of breast milk can be reduced, pairs of breast milk samples from the same mother with matching macronutrient content shall be pooled. Length of pooling intervals was set to alternating between Averaging macronutrient by combining breast milk samples with high and low macronutrient content: According to the selected macronutrient, pairs of samples  were pooled. Pooled samples were split into two of equal volume.(i) Combining breast milk samples with the highest macronutrient content: The three samples containing the highest amounts of the selected macronutrient were mixed and divided into equal samples containing a volume of 100 mL each. The remaining three or five batches were not included into further analysis (see discussion).(ii) The content and variation of fat, protein, carbohydrates, and energy were calculated for each of the resulting batches.n = 1 and was increased up to n = 996 with increments of n = 5. Cut-off criterium for efficiency was the minimum set size needed to reduce protein content below 10% variability.This simulation addresses the question whether random pooling of native breast milk samples will be able to reduce the variability of macronutrients and, if so, which would be the most efficient set size. For this purpose, a defined number of native breast milk batches were randomly drawn from the original data set of 3,338 batches, were pooled and the resulting content of protein, fat, and carbohydrates was calculated. For any given set size, this step of random allocation was repeated 1,000 times and mean and SD were calculated to serve as an estimate of the most likely distribution of nutrient contents after pooling. Set size started at This simulation uses partial lyophilization to up-concentrate the breast milk content. Separate analyses were done for protein, fat, and carbohydrates as the leading macronutrient to determine the amount of water to be removed to meet ESPGHAN recommendations at a h feeding volume of 150 mL/kg/d . Up-concDescriptive statistics for the macronutrient content of native breast milk and the different approaches to target fortification were calculated using R version 3.5.3 .n = 103), mean, standard deviation, median, interquartile range, quantile distance Q0.1-Q0.9, and boxplots were calculated  of the “Level 1 analysis.” Boxplots and descriptive statistics were generated.Level 2 analysis employed an interindividual analysis. Results from “Level 1 analysis” were used to assess interindividual differences . Level 2Overall, the fat, protein, and lactose content of native breast milk showed high inter- and intra-individual variation and a high range of macronutrient content or composition , the median fat content exceeded the recommended intake by ~0.8 g/100 mL  while me0.1-Q0.9) was 0.6 g/100 mL for protein, 1.5 g/100 mL for fat, 1.3 g/100 mL for carbohydrates, and 15 kcal/100 mL for energy. Due to the volume displacement, the use of liquid fortifier (#11b) decreased the variation of macronutrients  when compared to powdered fortifiers  was calculated (fortifier 8–10). For all three measurement schedules final macronutrient content of breast milk batches fortified with the corresponding optimized fortifier were then computed . MacronuFortifiers 9 and 10 shall serve as base fortifiers for target fortification with either one measurement per week or 3 measurements per week, respectively. Macronutrient levels decreased with increasing measurement frequency. Interestingly, they did not require the addition of fat. Since fat content was highly variable and exceeded recommendations in a significant number of breast milk batches routinely adding more fat seems to be quite unreasonable. As a consequence of this approach, administering any extra fat by single macronutrient modular is not required in ~40% of the feeds. Compared to fortifier 9, fortifier 10 added lower amounts of protein (1.3 vs. 1.5 g) and carbohydrates (0.5 vs. 1.0 g).Overall, target fortification was able to achieve ESPGHAN recommended intakes. Target fortification based on just one breast milk analysis per week provided a median macronutrient intake that met recommended levels. However, some residual day-to-day variation remained. A clinically significant reduction in macronutrient variation was found when target fortification was based on three or more measurements per week .Target fortification using low-fat/high-carbohydrate standard fortifiers (fortifiers #1 and #2) led to some milk samples exceeding the recommended carbohydrate content without extra modular macronutrients being added, while the use of high-fat/low-carbs fortifiers (fortifiers #6 and #7) resulted in fat levels that exceeded recommendations. In these samples target fortification was not able to reduce the macronutrient variation as well as to achieve recommended intakes. The fortifier optimized for three measurements (fortifiers #10) came the closest to ESPGHAN recommended intakes for three or more measurements. The highest precision and accuracy of nutrient intake was reached with daily measurements.The use of the liquid fortifier (#11) led to median fat (5.6 g/100 mL) and energy  levels that were above the recommended intake. The use of liquid fortifier also reduced the variability of macronutrient content due to volume displacement [see also last paragraph of chapter Commercially Available Multicomponent Standard Fortifier (Fortifier 1–7 and 11)] One measurement per week for target fortification achieved a lower variation when compared with powdered fortifiers.Detailed data about energy intake, protein per energy ratio as well as carbohydrate energy per non-protein energy are presented in In a next step, we were aiming to evaluate the quality of nutrient supply provided by the different fortification regimes. For this purpose, rather than assess the quality of each single nutrient intake separately, we focused on the effect of the composite intake on growth. Growth is a function of the intake of all three macronutrients therefore we applied an established growth model to estimate ELBW infant growth rates assuming a fixed intake of 150 mL/kg/d for all fortification approaches , 33–35. Applying standard fortification, most samples led to growth rates below the defined tolerance range of ±1 g/kg/d. The optimized standard fortifier, i.e., no breast milk analysis (fortifier 8) lead to adequate growth in about 30% of the feeds. Using target fortification, a higher number of fortified batches met the tolerance range as the number of measurements per week increased. About 40% were inside the growth range when applying one measurement per week. The percentage of appropriately fortified samples increased in increments of ~10% along with the number of measurements per week. The highest accuracy hit the optimized fortifier with one (fortifier 9) or three (fortifier 10) measurements per week of 87–93% .Combining samples with opposite extremes [see Methods section Simulation of Selection and Combination of Specific Native Breast Milk Samples (i)] based on one macronutrient  or on energy resulted in a decrease in variation of more than 50% for the selected macronutrient, while the other macronutrients showed a smaller decrease in variation. When samples were combined according to energy content, the variation for fat, protein and carbohydrates decreased by 47, 33, and 31%, respectively .For the second approach, i.e., pooling breast milk samples with the highest content of either protein or carbohydrates or fat [see Methods section Simulation of Selection and Combination of Specific Native Breast Milk Samples (ii)] increased the selected macronutrient content by 9–15%. However, when ordered by energy or fat, the average macronutrient intake improved for the other macronutrients by 9–13% . This apPooling of at least 150 random breast milk samples is needed to reduce the variation of macronutrients to below ±10% (~0.3 g). Various factors for partial lyophilization were tested by up-concentrating native breast milk samples to achieve recommended ESPGHAN intakes for macronutrients and energy . No lyopThis study confirms the presence of considerable intra- and inter-individual variation of native breast milk composition with no relationship between macronutrients. This challenges the ability of fortification strategies to achieve recommended intakes. While standard fortification with fixed doses increases the macronutrient content of native breast milk, it does not reduce its variation and the associated risk of unbalanced intakes. Only target fortification, based on at least two breast milk analyses per week in combination with an optimized standard fortifier , was able to provide macronutrient intakes that adhered to the recommendations of nutritional committees .Evaluation of milk analysis of 3,338 samples and eight commercially available fortifier shows that standard fortified breast milk meets nutritional requirements as suggested by ESPGHAN in <12% of all feeds. Based on our data, we suggest a “new” optimized standard fortifier in order to meet these recommendations [see chapter Optimized Standard Fortifier ]. The composition of this fortifier differs from current commercially available fortifiers. The optimized fortifier increases the number of appropriately fortified batches; however, 70% of feeds will still have a more or less unbalanced composition of fat, protein, or carbohydrates. In conclusion, standard fortification is not suited to reduce the significant macronutrient variation of breast milk for all preterm infants, and instead transfers the degree of imbalance to higher macronutrient levels , 30, 37.Target fortification reduces the high macronutrient variation of standard fortified breast milk and improves deficient macronutrient content depending on the frequency of native breast milk measurements per week: one measurement per week will allow fortified breast milk to reach the average recommended macronutrient intake, although some intra-individual variation remained. Two or more measurements per week decrease the variation significantly. The best performance was achieved by daily measurements, however, with significantly increased clinical workload. This finding is comparable to our recently published randomized controlled trial, which also suggested to perform two or more weekly breast milk measurements to meet recommended macronutrient intakes and reduce variation .The efficiency of target fortification depends on the composition of the standard product used as base fortifier . Target In this present study, we propose an optimized composition for base fortifiers to reach recommended intakes with feeding volumes of 150 mL/kg/d of milk for most of the batches. The macronutrient composition of these optimized base fortifiers was different if developed for standard  or for target  fortification. Neither base fortifier for target fortification should contain fat. Additionally, the composition of the base fortifier for target fortification was determined by the frequency of breast milk analyses. With three measurements per week, the optimized base fortifier had a lower macronutrient content than the fortifier based on one measurement per week. In an individualized approach with more frequent measurements such lower macronutrient levels are needed to prevent overfortification of native breast milk. Conversely, deficient samples will be corrected by individually adding modular single macronutrient products. In an ideal scenario, milk analysis and adjustment by target fortification would occur daily. Hence, the development of a general base fortifier for target fortification would be oriented to adequately fortify samples with higher macronutrient levels  and thus contain rather low amounts of non-protein components. Besides that base fortifiers would also focus on the important role to provide preterm infants with micronutrients, electrolytes, vitamins, phosphate, and trace elements. The required fine-tuning with macronutrients , could then individually be made by single macronutrient modulars only according to the breast milk analysis. Consequently, a modern fortification concept for use in clinical routine would aim to standardize intake on the basis of a biologically valuable base liquid with random composition effect by applying real-time component analysis followed by base fortification with (full) micro- and  macronutrients using an optimized base fortifier plus fine-tuning with three modular for protein, fat and carbohydrates.Our study demonstrated that the commercial liquid fortifier based on human milk provides higher amounts of fat than powdered standard fortifiers. Most of the samples fortified with the liquid fortifier had a higher fat content than recommended by ESPGHAN. However, from a nutritional point of view, the quality of fat from human milk sources cannot directly be compared to fat from non-human milk. The fat fraction of human milk has a particular composition and is constituted by specific fat globules. These fat globules contain glycerophospholipids, sphingolipids, cholesterol, and proteins , 38, whiIn the present study we were able to show that selective batching of corresponding breast milk batches and/or up-concentrating by partial freeze drying are not appropriate to reduce the variation in the composition of breast milk and to meet the needs of preterm infants. Two main factors limit the effectiveness of these approaches. First, there is only a weak correlation between fat, protein, and carbs . TherefoThese findings have implications for breast milk preparation. Our data show that on a large scale, like in a milk bank setting, batching of a high number of milk samples (>150) has a high likelihood of creating large volume batches with a similar composition and less macronutrient variation. However, even considering that our sample set might provide a higher variability due to early milk vs. mature milk that a milk bank would have in stock, batch sizes would exceed reasonable numbers. However, on an individual level, mixing, and batching of a small number of breast milk batches only from one mother is unlikely to make a difference of clinical significance. To attain an exclusively human milk diet from mother's own milk by freeze-drying is also infeasible: we were able to demonstrate that the lyophilized human milk concentrates generated in this study fail to provide recommended intakes. The degree of up-concentrating native breast milk to optimize protein intake would lead to a significant over-fortification (more than double) of fat and carbohydrates. Further, freeze-drying could negatively affect infant's intake of vitamins and micronutrients  and incrThis study suggests that individualized target fortification of breast milk is able to provide an adequate intake of nutrients at recommended intakes with reduced macronutrient variability. In contrast, current practice of standard fortification provides an uncertain, probably sometimes even unsafe intake of imprecisely defined amounts of macronutrients, unlike the administration of parenteral nutrition or medications which requires accurate dosage and where such practice of random composition would be considered as unacceptable. Unbalanced or excessively high or low intakes of macronutrients may only be recognized by growth failure or metabolic disturbances and hence there inherently is a delay in detection. However, an optimal macronutrient composition is needed to achieve desired body composition outcomes. Recent studies have demonstrated that optimized nutrition leads to higher percentages of fat free mass, which is associated with faster neuronal processing, larger brain size and improved motor development –46. Unban = 3,338 of n = 103 subjects), the daily sample collection for a minimum period of 15 days during the NICU stay, and the use of validated methods to precisely measure macronutrient content of breast milk  to be used in target fortification would improve the macronutrient intake and thus growth.kinderzentrum@klinikum-nuernberg.de.The datasets presented in this article are not readily available because restricted by the Study protocol and Research Ethics Board of McMaster University. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Research Ethics Board of McMaster University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CF and NR: conceptualization, methodology. HS, MR, and NR: formal analysis. GF and SF: milk analysis. GF, NR, and SF: data curation. EY, NR, and SF: writing—original draft preparation. CF, MR, NR, and SF: visualization. NR: supervision. CF: project administration. All authors writing—review and editing. All authors have read and agreed to the published version of the manuscript.This study was partially funded by Canadian Institute of Health Research, #MOP-125883.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The ancestors of cyanobacteria generated Earth’s first biogenic molecular oxygen, but how they dealt with oxidative stress remains unconstrained. Here we investigate when superoxide dismutase enzymes (SODs) capable of removing superoxide free radicals evolved and estimate when Cyanobacteria originated. Our Bayesian molecular clocks, calibrated with microfossils, predict that stem Cyanobacteria arose 3300–3600 million years ago. Shortly afterwards, we find phylogenetic evidence that ancestral cyanobacteria used SODs with copper and zinc cofactors (CuZnSOD) during the Archaean. By the Paleoproterozoic, they became genetically capable of using iron, nickel, and manganese as cofactors . The evolution of NiSOD is particularly intriguing because it corresponds with cyanobacteria’s invasion of the open ocean. Our analyses of metalloenzymes dealing with reactive oxygen species (ROS) now demonstrate that marine geochemical records alone may not predict patterns of metal usage by phototrophs from freshwater and terrestrial habitats. How early photosynthesizers managed oxidative stress remains relatively unresolved. Analyses of enzymes dealing with reactive oxygen species traces the evolutionary history of superoxide dismutases and finds evidence of CuZnSOD in the ancestor of all cyanobacteria, dating back to the Archaean. Although today the Earth’s atmosphere contains ~21% oxygen (O2), it was at least 105 times lower in the Archaean 4.0–2.5 billion years ago (Gya)3. Just how and when O2 first appeared as a byproduct of biological evolution-oxygenic photosynthesis - remains controversial, with estimates ranging from near the origin of life4 to 3.8 billion years ago (Ga)5 to immediately preceding the Great Oxidation Event (GOE)6, which is estimated to have begun 2.50–2.45 Ga8. Before this, O2 could have been produced by physical processes, such as photodissociation of water and carbon dioxide by UV light10, but is unlikely to have accumulated at appreciable levels. With the evolution of oxygenic photosynthesis came the potential to produce O2 on a much larger scale. Since O2 is highly reactive, early Cyanobacteria—the first producers of biogenic O2—likely experienced selective pressure, resulting in the evolution of more efficient antioxidants. Such effects have been documented in the photosynthetic machinery, which has been evolving strategies of dealing with reactive oxygen species (ROS) throughout its history11. Therefore, it is reasonable to assume that O2-generating organisms, such as cyanobacteria, would have co-evolved more efficient mechanisms of managing ROS as water oxidation proteins evolved.Oxygen is essential for complex life forms as it is used during aerobic respiration to create more energy per mol of substrate than other available electron acceptors12, and their evolutionary history can be elucidated with phylogenetic methodologies. Although peroxidases and catalases enhance the rate of removal of peroxides (such as H2O2 and R-O-O-H)13, SORs and SODs remove superoxide free radicals (O2.−)14. These O2.− are produced as a byproduct of photosynthetic and respiratory electron transport chains12 as well as extracellular processes on the cell surface15. They can also have beneficial roles in iron acquisition, cell signaling, and growth16, but if O2.− are allowed to accumulate inside the cell, they react with solvent-exposed 4Fe-4S clusters in proteins, including those required for amino acid biosynthesis17 and photosynthesis18, generating reactants of the Fenton reaction, which can ultimately lead to extensive DNA damage12. To balance the beneficial effects of O2.− with damage caused by over-exposure, organisms must maintain control over their abundance. Perhaps, it is for this reason that SODs and SORs have been found in all three domains of life—Eukarya, Archaea, and Bacteria14.Cyanobacteria remove ROS using carotenoids, α-tocopherol, and antioxidant enzymes, including peroxidases, catalases, superoxide reductases (SORs), and superoxide dismutases (SODs)2.− 19. Each has a unique 3D structure, amino acid sequence, and metal cofactor(s); either manganese (MnSOD), nickel (NiSOD), or a combination of copper and zinc (CuZnSOD)20. A fourth SOD enzyme shares its evolutionary heritage with MnSOD, but utilizes iron as its cofactor (FeSOD)21. All of these SODs are found within cyanobacteria23, but their locations within the cell differ. FeSOD and NiSOD are cytoplasmic27, whereas CuZnSODs from Synechococcus and MnSODs from Anabaena and Plectolyngbya are tethered to membranes30. The choice of which SOD isoform(s) and associated metal cofactor(s) are used by a given species could reflect its evolutionary heritage, function, and environmental history.Phylogenetic methodologies coupled with protein structural analyses have revealed that three different isoforms of SOD enzymes evolved independently of one another to remove unnecessary O31), and perhaps as many as or >3.22 Ga . During this time, cyanobacteria have diversified into a wide range of marine, freshwater, and terrestrial habitats35. Early phylogenetic studies on the evolution of SODs in cyanobacteria were limited by the availability of genomes but found that NiSODs are encoded in the genomes of planktonic marine strains (such as Prochlorococcus spp.), whereas MnSOD and FeSOD are more widespread36. CuZnSODs, while rare, are not restricted to any particular group23. The 3D structures required for metalloproteins to incorporate copper cofactors have previously been predicted to have arisen after those required to incorporate Fe- and Mn-utilizing proteins in the Proterozoic (~2.4–0.5 Ga), following the GOE37. Therefore, it would follow that cyanobacteria used FeSODs and MnSODs before CuZnSODs. Although genes encoding CuZnSODs and FeSODs/MnSODs are widely distributed in bacteria and eukaryotes, NiSODs are restricted to bacteria and may have appeared later in the evolutionary history of life19. Previous approaches aimed at determining metal usage in SODs have not considered the habitats where strains live or evolved. This is problematic because metal availability differs across oceanic and terrestrial environments39.The evolutionary record of cyanobacteria spans at least 1.88 Ga , CuZnSOD (sodC), FeSOD (sodB), and MnSOD (sodA) in the context of their habitat and metal cofactor availability through geological time. To do this, we construct phylogenies of all SODs encoded in the genomes of 15,899 bacteria. We also implement a phylogenomic approach and multiple molecular clock analyses to estimate when the crown group of cyanobacteria diverged from their closest non-photosynthetic relatives, the Vampirovibrionia41. By studying the timing of the divergence of cyanobacteria from their closest relatives, as well as their acquisition of SODs, we establish points in the Proterozoic and Archaean when these SODs were present in cyanobacteria.Here, we analyze when cyanobacteria acquired genes encoding NiSOD  of beta-, gamma- and alpha–proteobacteria as well as planctomycetes, bacteroidetes, actinobacteria, cyanobacteria, and firmicutes  live in saltwater habitats  encoding NiSOD’s maturation protease  do not  are all marine with small genomes. They include picocyanobacteria and endosymbionts living in larger marine tunicates, algae, and sponges . The 70 cyanobacteria which only use FeSOD and MnSOD live in a variety of marine, terrestrial, and freshwater habitats , whereas CuZnSOD is composed of 177 (range 103–236) and FeSOD/MnSOD of 200 (range 197–280)  phylogenies were constructed using the NiSODs, CuZnSODs, and combined Mn- and Fe-SODs from bacteria. If, in each case, all cyanobacterial proteins form a monophyletic group , this would indicate a single origin. Surprisingly, these phylogenetic analyses revealed that cyanobacterial NiSODs, CuZnSODs, MnSODs, and FeSODs are polyphyletic Fig. , suggest40 Fig. .sodN (NiSODs), five of sodC, and three to eight of Mn- and Fe-utilizing SODs  diversified from a protein resembling those of alphaproteobacteria  of each SOD isoform between cyanobacteria and other bacterial phyla: Two of ODs Fig. . A variePseudanabaena spp. and Gloeobacter spp.) share sodC genes which are monophyletic (PP 1) and closely related in the same way as the species are to one another  in seawater to form banded iron formations (BIF)54. Organisms would have responded by choosing more bioavailable metals where possible and evolving more efficient enzymes, such as SODs, to deal with the expansion of O2 into new habitats34.The advent of oxygenic photosynthesis had profound impacts on Earth’s climate, chemistry, and biota. It destroyed a methane greenhouse which kept the world warm42, in the neoarchaean between 2.5 and 2.6 Ga55, but our molecular clock analyses point to an earlier origin where the cyanobacterial lineage emerges in the Paleoarchaean between 3.3 and 3.6 Ga based on mean predicted ages   which predicts the rapid proliferation of these enzymes 3.1 Ga59; although differences arise in the dating of FeSOD and MnSOD between this study and that of ref. 59 (see below for discussion).Age estimates for the emergence of oxygenic photosynthesis are therefore fundamental to reconciling the biochemistry and use of trace metals. Previous estimates place the divergence of cyanobacteria and their closest relatives, vampirovibrionia2.− 14 and when production is particularly high (owing to bright light for example), SODs protect the photosynthetic repair mechanisms from damage61. Key parts of this photosystem repair mechanism (namely membrane-bound FtsH proteases which degrade damaged photosystem components) evolved with the advent of oxygenic photosynthesis in the Archaean62. Our phylogenetic analyses suggest for the first time that CuZnSOD, an antioxidant enzyme capable of protecting photosynthetic repair mechanisms from oxidative damage25, was also present in cyanobacteria in the Archaean, at least 2.9–2.6 Ga  produces OPseudanabaena spp. and Gloeobacter spp.  , were present in the Archaean, 2.7 Ga64. If proteins could incorporate Cu cofactors in the Archaean, then CuZnSODs could have been present at the root of cyanobacteria as a mechanism to deal with O2 generated by photosynthesis before it accumulated in the global atmosphere (at the GOE). This would have been particularly important for Mesoarchaean and Neoarchaean individuals living inside benthic mats66, where there was a smaller diffusion gradient to pull O2 out of the cells. As a result, higher concentrations of O2 accumulated, raising the potential for superoxide radical generation, particularly in the periplasm67. In the alternative (if unlikely) scenario where basal cyanobacteria acquire sodC via HGT ~ 0.95 Gya, Archaean cyanobacteria might not have used SOD to remove O2.− because we do not find any phylogenetic evidence for NiSOD, MnSOD, or FeSOD having been inherited from an ancestor present before the GOE  in the open ocean at the end of the Precambrian78. Although only speculative at this time, it is possible that the acquisition of NiSOD and its associated maturation protease assisted the invasion of cyanobacteria into pelagic marine habitats. Pelagic planktonic cyanobacteria are typically limited by P, Fe, and N (in cases of non-nitrogen fixers) owing to their distance from sources of riverine discharge79. As NiSOD is composed of fewer amino acids than other SOD isoforms  to track seawater composition through time (see ref. 39 for a review). With regards to the metal cofactors contained in SOD, their temporal trajectories have been reconstructed based on both thermodynamic considerations (e.g.83) and sedimentary records . However, there is a poor record of trace metal inventories outside of marine habitats, so it can be more difficult to establish links between metal availability and diversification of cyanobacteria living in terrestrial and freshwater habitats.It has been proposed that trace metal inventories of ancient marine sediments should reflect trace metal availability and correspond to the emergence of novel metabolisms and metalloenzymes85 and Cu86 in the water column. This may hint at either a strong biological control on water column cycling being established very early on or the possible complexation of these trace metals by organic ligands. Although the possible release of Cu through the oxidative weathering of sulfides in the lead up to, and following, the GOE86, may explain the emergence of CuZnSOD in the Neoarchaean, at this point there is a dearth of geochemical evidence to support this hypothesis, at least from a global first-order perspective. Despite this, existing chemical sediment compilations cannot preclude the possibility of local or transient increases in Cu, creating oases of bioavailability that stimulated the emergence of CuZnSODs.Our ancestral state reconstructions predict that a non-marine cyanobacterium could have acquired CuZnSOD 88. In turn, this would have created environmentally favorable conditions for a rise in oxygenic photosynthesis by cyanobacteria. This Neoarchaean to Paleoproterozoic decline in Ni is also reflected in a compilation of shale data, normalized to evolving upper continental crust .Past reconstructions of Ni in seawater based on BIF and syn-sedimentary to early diagenetic pyriteust Fig. . Interesust Fig.  that occust Fig. , which i91, or through the weathering of freshly emplaced large igneous provinces as has been proposed for phosphorous92. At present, the exact mechanisms and timing of higher Ni concentrations in Neoproterozoic shales require further investigation as it lags behind the observed increases in molybdenum, vanadium, and phosphorus93.Elevated Ni/Ti ratios during the Neoproterozoic presumably reflects a relatively greater Ni flux to the oceans from either increased oxidative weathering, similar to other sulfide bound metals such as molybdenum or vanadium at this time37. Alternatively, this has been proposed to reflect an intrinsic biological property of eukaryotic evolution rather than a sudden shift in Zn availability84. In the latter view, the early incorporation of Cu and Zn into SODs would reflect a lack of competition from the yet unevolved eukaryotes in the Archaean. Then in the late Mesoproterozoic, as eukaryotes began to become more abundant and dominate marine and terrestrial environments, they may have outcompeted cyanobacteria for Cu and Zn, providing an impetus for the emergence of other membrane-bound SODs, such as MnSOD—effectively providing a strategy for alleviating limitation imparted by competition with emergent eukaryotes. Although eukaryotes also utilize Mn, protein structures required to bind Mn have not been preferentially retained in their genomes94 and cyanobacteria, having evolved earlier than photosynthetic eukaryotes95, had plenty of time to develop an efficient Mn uptake system.Given the histories of these trace metals, one may ask whether it is availability driving the assimilation of Cu, Zn, Fe, Mn, and Ni into SOD metalloenzymes, or an inherent competition with other extant lineages at any given time. For instance, metalloenzyme fold superfamilies that incorporate Zn proliferate late and have been proposed to contribute to the delay in eukaryotic evolutionsodN incorporation into the cyanobacterial genomic repertoire coincides with Earth’s second planetary oxygenation3 and the emergence of planktonic marine cyanobacteria35. Therefore, we suggest that cyanobacteria began assimilating nickel into SODs to invade Neoproterozoic planktonic communities as Neoproterozoic oxygen levels rose.Later, in Neoproterozoic marine habitats, cyanobacteria began supplementing their ROS defense mechanisms with a new and smaller SOD isoform (NiSOD) that requires less phosphorus and nitrogen to manufacture. This timing of https://www.ncbi.nlm.nih.gov/refseq/) in 2018. They were searched for genes encoding NiSOD, CuZnSOD, FeSOD, and MnSOD using the basic local alignment search tool (BLAST) for proteins (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with a word size of six, gap opening penalty of 11, gap extension penalty of 1, and e value cut off of <1 × 10−5. Query sequences are detailed in Supplementary Table https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) 40.Cyanobacterial SOD diversity was assessed by sampling 153 additional genomes  were aligned using MAFFT77. Each protein and ribosomal RNA were aligned using MAFFT96 and concatenated using an alignment viewer . Gaps were removed if present at the same position in ≥80% sequences and ML methodology was implemented in IQ-TREE v1.6.146 to estimate the cyanobacterial phylogeny. Each protein and rRNA was characterized under a unique partition with -SP to account for heterotachy and appropriate substitution models identified by ModelFinder97. Ultrafast bootstrap values were calculated to measure support for branching relationships by resampling sites within partitions 1000 times98.An evolutionary tree of cyanobacteria was generated from a data set including SSU rRNA (1687 nucleotides), LSU rRNA (3387 nucleotides), and 136 core proteins . Each protein is encoded by an orthologous gene, which is conserved among cyanobacteria and involved in a key cellular function, such as information processing, metabolism, or cellular processes. For a full description of these genes, see previous papers99 (CAT-GTR) to account for different rates of evolution between distant sites of the molecule. Divergence times were estimated in phylobayes 4.145 using uncorrelated gamma multipliers,49, a birth-death prior on divergence times, and root prior chosen from a Gamma probability distribution with mean 3060 and standard deviation 404. As a result, 97% of the prior distribution specified that the ancestor of all vampirovibrionia and cyanobacteria originated after the end of the late heavy bombardment 3.9 Ga101.Bayesian relaxed molecular clocks were implemented using the topology described above. This topology was fixed, and ages estimated based on predicted mutation rates for ribosomal RNA . Substitutions were modeled using a flexible general time-reversible model inferred from the alignment and a Dirichlet process prior103, akinete-forming cyanobacteria 1.6–1.888 Ga105, and apical cells of cyanobacteria 1.7–1.888 Ga106. These fossil constraints were supplemented with evidence dating the appearance of eukaryotic hosts of endosymbiotic cyanobacteria. For example, Richelia species diversify before their diatom host, named Hemiaulus107, appeared 110 Mya108, and UCYNA species diversify before their prymnesiophyte host, named Braarudosphaera bigelowii, appeared 91 Mya109. Geochemical evidence was used to constrain cyanobacteria to first diversify before the Great Oxygenation Event of 2.32 Ga110, but after either; (a) 3 Ga when molybdenum oxides document the first “whiffs” of atmospheric oxygen56; or (b) 2.7 Ga based on the earliest fossilized evidence of cyanobacterial stromatolites111. Soft bounds were applied throughout to allow 5% of the prior probability density to fall outside of the specified minimum and maximum ages.We also implemented microfossil calibrations as follows; filamentous cyanobacteria >1.9 Gaeffsize > 50, reldiff < 0.3). Chronograms and divergence times were calculated from a single representative chain using Readdiv (in Phylobayes) with the same burn-in (25% of mean chain length) and sampling frequency (1 in every 10 points) as Tracecomp.Models were considered complete when four replicate independent chains converged. This was tested by estimating effective sample sizes and relative differences using Tracecomp . Character states were coded as either “marine” or “non-marine” . This normalization accounts for secular changes in the composition of Earth’s continental crust that reflect the emergence, growth, and subsequent differentiation of continental crust from the Archaean through the Phanerozoic. The evolution of continental crust influences the relative availability of trace and major elements weathered from land to the oceans, and thus the normalization removes this temporal variability, facilitating a comparison of Ni/Ti ratios in shales through time. Mean values for each time bin were bootstrap resampled (n = 10,000; Fig. From a database of >4000 literature shale analyses spanning the Archaean to modern, 1584 had both Ni and Ti data available and are used here to reconstruct the trajectory of Ni in seawater (Supplementary Data Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Utilization of cell free DNA for diagnosing and monitoring patients with biliary tract cancers is emerging and promising. The strength of the present study is in its description of a novel approach using bile rather than blood or tissue samples, which is particularly relevant in biliary tract cancers. This paper largely serves as a proof of concept that ctDNA from bile is potentially feasible.KRAS somatic mutation. KRAS mutations were identified in the bile ctDNA of 20 of 42 (48%) patients. Patients with mutant KRAS showed significantly worse survival than those with wild-type KRAS . There was 80.0% mutational concordance between the paired bile ctDNA and FFPE samples, and 42.9% between the plasma and FFPE samples. On transcriptomic sequencing of one set of paired bile and FFPE samples, expression level of KRAS-associated signaling oncogenes in the bile and tissue samples showed a strong positive correlation . Liquid biopsy of bile reliably detect mutational variants within the bile ctDNA of BTC patients. These results suggest that bile is an effective biopsy fluid for ctDNA analysis.Although liquid biopsy of blood is useful for cancer diagnosis and prediction of prognosis, diagnostic and prognostic value of ctDNA in bile fluid for BTCs are not clear yet. To determine whether liquid biopsy for circulating tumor DNA (ctDNA) can replace tissue biopsy when assessing somatic mutations in biliary tract cancers (BTCs). Bile samples were obtained from 42 patients with BTC. Matched formalin-fixed paraffin-embedded (FFPE) samples were obtained from 20 of these patients and matched plasma samples from 16 of them. Droplet digital PCR (ddPCR) was used for detection  Biliary tract cancers (BTCs), which include gallbladder cancer (GBC), ampullary carcinoma (AC), and extrahepatic cholangiocarcinoma (eCCA), are rapidly growing tumors that are usually lethal ,2,3. TheLiquid biopsy can be useful for cancer detection, monitoring, and management. Fragmented DNA circulates in the cell-free component of whole fluids. The cell-free circulating tumor DNA (ctDNA) in the blood is DNA released from apoptotic, circulating, or living tumor cells. ctDNA is about 140 nucleotides long and has a half-life of about 1.5 h. The analysis of ctDNA provides a noninvasive way to assess the genetic profiles of cancers in real time . In healKRAS is frequently mutated in largely diffuse tumors, such as pancreatic cancers, colon cancers, and BTCs [KRAS mutations in BTCs is 9–40% [KRAS mutation has been associated with perineural invasion, advanced stage, and poor prognosis in these patients [KRAS mutation in codon 12 is especially associated with worse survival in patients with eCCA [KRAS mutations is associated with resistance to several targeted treatments, including tyrosine kinase inhibitor and anti-epidermal growth factor antibody treatments [KRAS mutations may be important in targeted approaches to treatment. Considering the invasiveness and low accuracy of tissue biopsy in BTCs [KRAS changes in ctDNA, may be valuable.Recent molecular analyses have identified several markers of poor prognosis and the activation of oncogenic pathways. and BTCs ,4,17,18.is 9–40% , and KRApatients . These mpatients . The foueatments ,23. TherKRAS mutations are frequent and have prognostic implications, we focused on the detection of these mutations in bile fluid. In this study, we used droplet digital PCR (ddPCR) to detect somatic mutations in KRAS in liquid biopsy samples to assess whether this technique can replace tissue biopsy as a diagnostic and prognostic tool for BTCs. For that, we compared the KRAS mutations detected in tissue, plasma, and bile samples. In addition, expression of KRAS-associated signaling oncogenes with next-generation sequencing (NGS), and analyzed correlations of expression among bile, and paired tissue and plasma.Few studies have assessed the diagnostic and prognostic value of ctDNA in bile fluid for BTCs. Because This prospective study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (DSMC 2018-06-054) and included the secondary use of human-derived materials. We obtained bile fluids from 46 individuals, including 42 patients with BTCs , and 4 patients with benign biliary tract disease (CBD stones) as a control, at Keimyung University Dongsan Hospital between August 2018 and March 2020. The diagnosis of BTC was based on a tissue sample obtained by surgical resection, needle biopsy or cytology with endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic biliary drainage (PTBD). When eCCA patients had isolated CCA without metastatic lesions and their general status was appropriate for surgery, curative surgical resection was performed. However, when patients had metastatic lesions or refused surgical resection, we administered chemoradiotherapy or palliative therapy . The tumg for 10 min at 4 °C to obtain the supernatant (bile). Each pellet was resuspended in chilled phosphate-buffered saline (2-fold), and then centrifuged again 16,000× g for 5 min at 4 °C to obtain the bile pellet, as described in the protocol of Abi Zabron  [The collected bile fluid was centrifuged immediately at 16,000× England) . All sam® cfDNA XS, NucleoSpin® FFPE DNA, and NucleoSpin® Tissue XS kits were purchased from Macherey-Nagel . The ddPCR™ KRAS Screening Multiplex Kit and ddPCR™ KRAS G12/G13 Screening Kit were purchased from Bio-Rad Laboratories . Ethanol and all other chemicals were obtained from Sigma .The NucleoSping for 15 min at 4 °C to obtain plasma. Plasma was stored at −80 °C .Blood from patients was collected preoperatively by vacutainer tubes containing heparin as an anticoagulant and was centrifuged 25,000× g for 10 min at 4 °C to obtain the supernatant (bile), as described in the protocol by Abi Zabron  [® cfDNA XS Kit, according to the manufacturer’s instructions. The DNA was analyzed on an Agilent 2100 Bioanalyzer.To separate the bile for ctDNA analysis, the collected bile fluid was centrifuged immediately at 16,000× England) . All of ® FFPE DNA Kit, according to the manufacturer’s instructions. The DNA was stored at −20 °C before analysis.FFPE samples were obtained from 20 patients who underwent surgical resection or endoscopic biopsy. Histological examinations confirmed that these samples contained at least 30% tumor cells. We extracted the DNA from sections (5 mm thick) of the FFPE samples (15 mg) with the NucleoSpinKRAS Screening Multiplex Kit and ddPCR™ KRAS G12/G13 Screening Kit (Bio-Rad Laboratories) on the Bio-Rad QX200 ddPCR system. A two-dimensional (2D) fluorescence amplitude plot generated with the Quanta Soft™ software showed the wells of mixed mutant and wild-type samples. 2D plots were constructed from the results of the ddPCR KRAS multiplex assays applied to the ctDNA from the bile samples from the no-template control (NTC), the BTC patients, and the control patient. The black cluster on the plot represents the negative droplets; the green cluster represents the droplets that were positive for wild-type DNA only; the blue cluster represents the droplets that were positive for mutant DNA only; and the orange cluster represents the droplets that were positive for both mutant and wild-type DNA . The amplification products were detected with a C1000 Tough Thermal Cycler (Bio-Rad Laboratories). The reactions were initiated by incubation at 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s, 55 °C for 60 s and 98 °C for 10 min. The concentration of DNA was calculated from copy number variations . The fragment size distributions of the bile ctDNA and FFPE DNA were analyzed with an Agilent 2100 Bioanalyzer . The fragment size of the bile ctDNA and FFPE DNA were analyzed with an Agilent 2100 Bioanalyzer using a high sensitivity DNA kit (Bio-Rad Lab).DNA fragmentation enables optimal accuracy by separating tandem gene copies, reducing sample viscosity, and improving accessibility. ddPCR was performed on DNA libraries with the ddPCR™ ® sequencing platforms), and its quality was confirmed with the TapeStation DNA Screen Tape D1000 . The indexed libraries were then submitted to Illumina NovaSeq , and paired-end (2 × 100 bp) sequencing was performed by Macrogen Incorporated .In one patient (CA34), for whom bile, tissue, and plasma samples were available, an mRNA-Seq data analysis was performed with NSG to directly compare the gene expression profiles detected in the three biological samples. The total RNA concentrations were calculated with a Quant-iT™ RiboGreen™ RNA Assay Kit . To determine the DV200 value (% of RNA fragments >200 bp), the samples were run on a TapeStation RNA Screen Tape system  The total RNA (100 ng) was used to construct sequencing libraries with a SureSelectXT RNA Direct system , according to the manufacturer’s protocol. Briefly, total RNA was first fragmented into small pieces with divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA with reverse transcriptase and random primers, followed by second-strand cDNA synthesis. These cDNA fragments underwent an end repair process, with the addition of a single ‘A’ base, and were then ligated to adapters. The products were purified and enriched with PCR to create the cDNA libraries. To capture the human exonic regions, we used an Agilent SureSelectXT Human All Exon V6 + UTRs Kit , according to the standard SureSelect Target Enrichment protocol . The cDNA library (250 ng) was mixed with hybridization buffer, blocking mix, RNase blocker, and 5 µL of the SureSelect XT Human All Exon V6 + UTRs capture library. Hybridization to the capture baits was performed at 65 °C with the heated thermal cycler lid option at 105 °C for 24 h on a PCR thermal cycler. The captured library was then washed and subjected to a second round of PCR amplification. The final purified product was quantified with quantitative PCR (qPCR), according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Standards Kits for IlluminaThe RNA isolated from each sample was used to construct a sequencing library with a SMARTer smRNA-Seq Kit (Illumina Inc.), according to the manufacturer’s protocol. Briefly, the input RNA was first polyadenylated to provide a priming sequence for an oligo (dT) primer. cDNA synthesis was primed with the 3′ smRNA dT Primer, which incorporates an adapter sequence at the 5′ end of each RNA template, to which it adds nontemplated nucleotides. These nucleotides were bound by the SMART smRNA Oligo, enhanced with the locked nucleic acid (LNA) technology for greater sensitivity. In the template-switching step, PrimeScript reverse transcriptase (RT) used the SMART smRNA Oligo as the template for the addition of a second adapter sequence to the 3′ end of each first-strand cDNA molecule. In the next step, full-length Illumina adapters (including the index sequences for sample multiplexing) were added during PCR amplification. The forward PCR primer bound to the sequence added by the SMART smRNA Oligo, whereas the reverse PCR primer bound to the sequence added by the 3′ smRNA dT Primer. The resulting cDNA molecules in the library included the sequences required for clustering on an Illumina flow cell. The libraries were gel-purified with BluePippin, and validated by checking their sizes, purity, and concentrations on an Agilent Bioanalyzer. The libraries were pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2500 instrument to generate 51-base reads. Image decomposition and the calculation of quality values were performed with the modules of the Illumina pipeline.We preprocessed the raw reads from the sequencer to remove low-quality and adapter sequences before their analysis and aligned the processed reads to the Homo Sapiens genome assembly (GRCh38) using HISAT v2.1.0 . HISAT u2 test for categorical variables. Overall survivals were calculated using the Kaplan–Meier method. Pearson’s correlation coefficient was used to measure the statistical relationship between two continuous variables. Cohen’s kappa coefficient was performed to evaluate the concordance between plasma, bile and FFPE. A difference with a p value of <0.05 was considered statistically significant.All statistical analyses were performed with SigmaStat statistical software . Continuous values for results of statistical analysis were expressed as the means ± SD. For comparisons between two groups, a Mann–Whitney U-test was used for continuous variables and a χKRAS are summarized according to sample type  in p = 0.284; p = 0.313; KRAS mutation in bile ctDNA showed significantly worse survival than those with wild-type KRAS   C. These KRAS duplex assay was applied to the bile, plasma, and tissue samples. Histograms of the NTC, control, and BTC mutations in bile are shown in KRAS multiplex in bile, FFPE, plasma were displayed in To observe the length of DNA fragments, we first compared the size distributions of the ctDNA molecules in the bile, plasma, and FFPE. The size of the DNA fragments was analyzed with an Agilent 2100 Bioanalyzer . As shown in KRAS DNA (copies of mutant DNA per droplet) were estimated from a Poisson distribution. The mean concentration of mutant DNA in the bile ctDNA from BTC patients  was about 10-fold higher than that of the control patient with a CBD stone  of the BTC patients (N = 42) with a dual multiplex assay, and included G12D, G12V, G13D, G12A, G12C, G12R and G12S. KRAS allele frequencies were displayed in KRAS mutations in their bile ctDNA, the frequencies of G12D and G12V were 20/20 (100%) and 9/20 (45%), respectively  (16/20)  was about 5-fold higher than in the control (4100 copies/mL). Therefore, we set the cut-off value for opies/mL . Among 1 or G12V C. We als (16/20) D. These KRAS ctDNA in plasma was very low and KRAS mutations and KRAS allele frequencies were only detected in three samples (18.8%), with a cut-off value of 60 copies/mL (p = 0.825).However, the concentration of mutant opies/mL . Among tKRAS gene expression was determined with RNA-Seq by Expectation-Maximization (RSEM) (v1.3.1) with the options: estimate, rspd; seed-length, 15; strandedness, forward. Representative electropherograms for bile, the paired tissue, and plasma mRNAs are shown in KRAS-associated signaling oncogenes, including RAS and RAF, among the three groups , as did those in the bile and plasma samples  and tissue and plasma  . These data suggest that both gene expression and mutations can be detected in bile and that bile reflects the gene expression profile of the BTC tissue.In one patient for whom bile, plasma, and tissue samples were available, transcriptomic sequencing was performed to verify the overall mDNA expression levels in the bile, tissue and plasma. We checked the template size distribution by running the DNA samples on an Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip (RNA fragments >200 bp). Weakly expressed mRNAs, with <10 transcripts per million read, in the bile, tissue, and plasma were excluded from the analysis. The reference gene annotation for Homo Sapiens  was retrieved from the National Center for Biotechnology Information (NCBI). The profile of e groups . The Vend tissue B. A corr< 0.001) C. These Endoscopic or percutaneous tissue biopsy for the diagnosis of BTCs is invasive and its accuracy varies (50–90%) ,29. TherKRAS, was detected at the same levels in the bile and paired tumor tissues from patients with BTCs. It means that bile accurately reflects the biological and oncological properties of the tumor tissue. Clinically, mutant KRAS transcripts were frequently detected in bile associated with advanced-stage (III/IV) tumors, and those patients had significantly worse survival rates than patients with wild-type KRAS transcripts. Shen and colleagues showed that although some configuration and effects are similar to our invention, only NGS is used and A146V, 136M and G12D are described among the KRAS mutation, and no description of the prediction of the prognosis of BTCs [KRAS mutations have a role as biomarkers of prognosis in BTC patients. Furthermore, ctDNA monitoring can provide a real-time insight into the response to treatment and may guide therapeutic adaptation in the future.In the present study, we have shown that liquid biopsy of bile, analyzed with ddPCR, can be used for the detection of DNA mutations. In addition, with transcriptomic sequencing, the expression of many genes encoding oncogenic signaling proteins, including  of BTCs . TherefoKRAS mutations was just 18.8% for plasma ctDNA compared with 48.0% for bile ctDNA. Moreover, the concordance between the KRAS mutations detected in bile and tissues (80.0%) was higher than the concordance between those in plasma and tissue (42.9%). Therefore, bile is a better biofluid than blood for the analysis of ctDNA in BTC patients, and in this study, we have demonstrated this superiority of bile biopsy in BTC patients for the first time. In the context of obstructive jaundice caused by BTCs, the exposure of the biliary epithelium and cancer cells to bile can be extensive, so the concentration of ctDNA derived from the tumor tissue can be high. However, we could not use fresh plasma in our comparison, and it is possible that the use of frozen plasma negatively affected our results. Because no other study has compared bile and plasma for the detection of ctDNA, a further comparative study of bile and plasma is required. In addition, there are a few mutant cases with close value to cut off values  and the concordance between tissue and plasma results (25–79%) was not high ,38. In tf values . In thesThis study had several limitations. Although it had the largest number of patients yet used to study bile ctDNA, the number of cases included was still insufficient for statistical validation. Because it was retrospective study and not all patients underwent tissue biopsy, we could get FFPE and plasma sample from some patients. Therefore, we must confirm our findings with a larger number of patients. Furthermore, the invasive procedure of endoscopic retrograde nasobiliary drainage via ERCP or PTBD is required to obtain bile fluid. However, in cases of obstructive jaundice caused by BTC or benign biliary stricture, biliary decompression is often required. Differentiating BTCs from benign biliary stricture is still clinically challenging, so liquid biopsy using bile may have clinical utility.KRAS variants for the diagnosis and prognosis of BTCs. The use of bile biopsy may lead to more rational, personalized, targeted therapeutic approaches to BTCs in the future.ctDNA is abundant in bile samples, and somatic variants of oncogenes in bile ctDNA can be detected with ddPCR. The high mutational concordance between bile and tumor tissue indicates that liquid biopsy to extract bile ctDNA can be used to detect


Cornu aspersum. It relies on protecting overwintering specimens with Lutrasil frost cloth (LFC). This approach yielded elevated mortalities in Romanian snail farms. We aimed to develop a simple and effective system for protecting overwintering C. aspersum adults in colder climates. This three-year, three-phase experiment investigated selected behavioral aspects and thermal protection efficiency of different protective structures. Mature gastropods in preparation for hibernation exhibited a significant preference for wood and ridge-tile micro shelters. Soil texture significantly influenced the burrowing behavior, but not the burrowing depth. The structure soil/LFC/straw/10-cm air cushion/high-density polyethylene (HDPE)—the “sandwich” system—was selected to be used as a protective system. Under farm conditions, adult snails tended to hibernate clustered together, attached to the lower surface of micro shelters. The “sandwich” system coupled with using ridge-tile/wood micro shelters resulted in significantly higher survival thanthe sole use of LFC. Predator occurrence appeared to exert a minor effect on snail survival. These data render the “sandwich” system a potential solution for overwintering C. aspersum breeders in colder climates.The Italian semi-intensive (ISISF) technology is widely used for rearing the Mediterranean snail, C. aspersum snails in Romanian snail farms. This three-year study aimed to develop a simple, but effective system for protecting breeders in colder climates. (2) Methods: The first phase investigated the (pre)hibernal burrowing behavior and the overwintering habitat choice. Protective structures based on straw, LFC, and/or HDPE were tested at pilot level (no snails). The most suitable system was applied under farm conditions. (3) Results: Wood and ridge-tile micro shelters were significantly preferred to corrugated iron micro shelters. Burrowing specimens acted as shallow-burrowers, and this behaviorwas significantly more common for looser soils. All pilot systems displayed significantly higher thermal protection efficiency compared to the sole use of LFC. The balance between straw moistening and thermal protection favored using structure soil/LFC/straw/10-cm air cushion/HDPE. Its use yielded significantly higher survival compared to the sole use of LFC. Most hibernating snails clustered together in large groups, attached on the lower surface of micro shelters. Predator occurrence appeared to marginally affect overwintering survival. (4) Conclusions: The “sandwich” system could be an effective solution for overwintering mature C. aspersum snails in colder climates.(1) Background: Hibernation in pens covered with LFC was associated with high mortality of  In the last decades, small animal farming has attracted increasing research and economic interest as a potential alternative to the husbandry of large animal farms . Since rearing small animals requires less water, space, and financial investment while also generating lower amounts of methane, nitrous oxide, and carbon dioxide than large livestock farming, these farms are more environmentally friendly and easier to implement and manage. Taken together with the reorientation of human nutrition towards a healthy diet, these advantages have encouraged the development of more sustainable, safer, and less costly farming alternatives, including the production of animal proteins from nontraditional farmed animals, such as terrestrial snails or insects .Cornu aspersum , being the main species of commercial interest. Native to the Mediterranean Basin and parts of Western Europe [C. aspersum easier, profitable, and less risky [Used as food since prehistoric times, the snail meat is highly prized in the modern diet due to its high content of proteins rich in essential aminoacids 12.9%), low content of fat , and elevated content of minerals  .9%, low ,4,5. Rean Europe . This reC. aspersum using the Italian semi-intensive snail farming (ISISF) system is based on the characteristics of the life cycle of this species, with the snails being bred, grown, and fattened on open pastures of fresh vegetables in free-range pens in a natural environment [The rearing of ironment . The breironment .2) is used to protect C. aspersum during hibernation [C. aspersum snails during the winter of 2005/2006 have exceeded 70% in most snail farms from Romania using the standard ISISF system [Driven by a significant share of economically underexploited arableareas and low labor costs compared to the Western European countries, this method of snail farming has enjoyed significant growth in the former communist countries of Central and Eastern Europe during the first decade of this millennium ,8. For eF system ,12. ThisF system .C. aspersum is a partially freezing tolerant species [C. aspersum can burrow itself into soil or leaf litter to avoidcold winter weather [Like other ectotherm animals, terrestrial gastropods spend the winter in hibernation—a physiological status of obligate dormancy during which metabolism decreases to subsistence level . Althoug species , for whi species ,17,18. T species . However weather ,19, but  weather ,21, yet C. aspersum, during the winter months. We first tested different types of protective structures built from materials with good thermal insulation properties that were easily available and cost-effective, more precisely high-density polyethylene (HDPE), straw, and LFC [Here, we aimed at developing a simple, cost-effective, but efficient system for protecting mature brown garden snails,  and LFC ,23,24. T2 each were fenced inside a breeding pen from a snail farm located in the village of Peciu Nou . After collecting the snails inside them, corrugated roofing sheets (rectangles of up to 0.25 m2), ridge tiles, or wooden pallets (325 × 265 × 70 mm) were put into each enclosure before populating it with 50 adult specimens of C. aspersum. The snails were collected after three weeks to determine their preference for different types of micro shelters.On 4 October 2004, three areas of 2.5 m2 each were randomly fenced inside the breeding pens of each farm, and the gastropods inside were transferred to other pens. Micro shelters  were then placed on the ground to cover at least half of each fenced area. After being populated with 100 adult individuals on 19 October 2005, the enclosures were covered with LFC (23 g/m2). The snails were collected on 1 December 2005.The burrowing behavior was investigated in two snails farms with different soil textures, that is a commercial heliciculture farm from the village of Urecheşti  having a medium-sandy clay soil; and another farm, located in the village of Banloc  and having medium clay soil. Three areas of 5 m2, and the protected surface was 10 m2 (5 × 2 m). During this one-week experiment, the inside and outside temperatures were measured daily with an electronic thermometer at 3 am. Measurements were performed at 5 cm above the soil surface for three points, i.e., both extremities of the structure and the middle of the structure. The average temperature during a 15 min. interval was recorded for each point, and only the mean value was used for statistical analysis.This study was conducted in March 2006 in the village of Sâmbata de Sus . Being located at the foot of the highest mountain peak from Romania , this site has cold to very cold winters, with frequent temperatures below −15 °C [C. aspersum. For example, this gastropod species is active at temperatures between 7 °C and 21 °C and an elevated humidity of 75–90% [C. aspersum exhibits a moderately enhanced cold hardiness concerning hibernation, and it cannot survive long periods of frost [C. aspersum snails. This parameter was estimated empirically by taking a bundle of straw out and twisting/squeezing it in the hand. The following classes of straw moisture were considered: (i) completely dry straw; (ii) slight straw moistening; (iii) moderate straw moistening; (iv) high straw moistening (wet straw).Apart from temperature, moisture levels are important for the viability of f 75–90% ,9. Underf 75–90% ,8,11. Imof frost ,15, suchof frost . Since pof frost , we consThe concept developed based on data derived from the pilot phase  was tested under realistic field conditions in three snails farms located in Cugir , Ezeriş , and Muntenii de Sus . These farms were populated with mature snails aged 12–16 months between May and June 2006. The initial herd was purchased from the International Snail Farming Institute . Following the standard workflow of the ISISF system, surviving specimens were transferred into newly established breeding pens about three months after populating the farm (from July to September 2006), with juveniles remaining in the first established breeding pens.C. aspersum snails. This reference enclosure was covered with LFC (23 g/m2) during the winter months.The “sandwich” system was used only for protecting the latter pens. It was installed in October/November 2006 when night temperatures were constantly below 5 °C for three consecutive days, and mature snails were observed starting to prepare for hibernation. Before this activity, the vegetation in the pens was cutoff to a height of 10 cm. The protective system was dismantled in March/April 2007 on dry weather when night temperatures constantly remained above 5 °C for at least four days. The thickness of the wheat straw layer was 10 cm for the farms located in Cugir and Ezeriş and 15 cm for the farm located in Muntenii de Sus. The rationale behind this was that the latter farm was in Moldova, a region influenced by Scandinavian-Baltic weather and with colder winters compared to the two former sites . At thisAfter dismantling the protective system, the breeding pens were watered daily for three consecutive days to promote spring arousal. All snails  were collected, and the living specimens were transferred into new breeding pens. The overwintering success was assessed based on the survival rate. Survival was expressed as a percentage and was calculated as the ratio of the number of living snails post-hibernation to the number of living specimens post-hibernation. We assigned a probable death cause to all collected shells. We did not know the actual date and cause of death but estimated them using the following criteria: (i) presence of the body inside the shell; (ii) extent of the weathering of shell nacreous interior after death; (iii) presence of shell smashed or with holes in the spire.We also determined the thermal efficiency of the “sandwich” system. Measurements were performed in triplicate within a breeding pen from the commercial heliciculture farm in Muntenii de Sus. The measurement protocol was similar tothat used during pilot testing. Measurement time points were chosen to reflect the thermal behavior of the “sandwich” system under both cold weather  and mild weather conditions .C. aspersum in outdoor snail farms.Throughout this time, the (pre)hibernal behavior of mature snails was monitored in six other heliciculture farms. Although investigating survival of juveniles and their (pre)hibernal behavior were not within the scope of the present work, general observations were, however, made. Taken together, these latter data should provide us with a more detailed understanding of behavioral aspects of (pre)overwintering 2 test on a 3 × 2 contingency table to determine the differences in the distribution of these classes across different types of micro shelters. In the case of significant results, posthoc analysis with Chi2 tests based on 2 × 2 contingency tables were applied to find which pairs of cells are significantly different. Paired comparisons with Chi2 tests based on 2 × 2 contingency tables were next run to assess the effect of soil texture on burrowing behavior characterized across three distinct parameters. To this end, the snails were first categorized into burrowing specimens and non-burrowing specimens. The burrowing gastropods were next classified into individuals burrowing within the top 5 cm soil layer and individuals burrowing deeper into the soil. After this, the non-burrowing snails were split into specimens attached to the lower surface of micro shelters and specimens lying on the ground (unattached).The snails were stratified into specimens attached to the lower (inner) surface of the micro shelters and unattached specimens. We conducted a ChiC. aspersum adults protected overwinter with the “sandwich” system  and those protected using only LFC (Muntenii de Sus) by conducting a Chi2 test on a 4 × 2 contingency table. In the case of significant differences, posthoc analyses were performed with Chi2 tests based on 2 × 2 contingency tables against the later variable. Finally, we applied a Chi2 test based on 4 × 4 contingency table on the distribution of shell condition classes to investigate differences in potential death causes across different locations. For significant differences, the frequencies observed for the reference pen were used as a benchmark for post hoc comparisons with Chi2 tests based on 2 × 2 contingency tables. All statistical analyses were performed using Statistica version 8 . In all of the cases, a p value less than 0.05 was considered significant. To reduce the error in approximation, we adjusted the values of Chi2 according to Yates’s correction for continuity and applied post hoc Wilcoxon tests with Bonferroni correction.A Friedman test was run on temperature data measured at a pilot level to identify the ability of different protection structures to preserve the soil thermal inertia during wintertime. Post hoc Wilcoxon tests against the outdoor temperature were conducted in the case of significant differences. The thermal efficiency of the protection system selected to be used was next analyzed under realistic field conditions. A Wilcoxon test was performed to compare the inside and outside air temperatures. Next, we determined differences in survival rates of C. aspersum snails for different types of micro shelters are shown in 2 test, p < 0.001). Post hoc testing revealed a significantly higher tendency of snails to avoid corrugated iron as a shelter when compared to ridge tiles and wooden pallets  were recorded throughout the experimental period, except for the second time point when moderate snowfall occurred. Temperatures measured at different time points inside and outside protection systems are given in p = 0.001). Post hoc analyses showed that temperatures inside all protective systems were significantly higher than the ambient temperature . Outdoor and indoor temperatures determined at selected time points are illustrated in Unlike pilot testing conducted in the village of Sâmbata de Sus, the area protected during this stage in the village of Muntenii de Sus was equal to the surface of a breeding pen . Post hoc testing revealed significantly higher survival rates for pens protected using the “sandwich” system versus the pen protected using only LFC . Post hoc analysis revealed significant differences when comparing the enclosures protected with the “sandwich” system to those protected using LFC hibernation, which until now was mainly related to the ethology of wildpopulations ,32,33,34C. aspersum, starts to prepare for hibernation [In response to mixed action of low temperature and decreasing photoperiod, the brown garden snail, ernation , with soernation . Here, mernation .C. aspersum adults in preparation for overwintering can display burrowing behavior. Soil texture significantly influenced this behavior, with the percentage of burrowing gastropods being two-fold higher in the case of looser soil. The most plausible explanation is that these soils provide easier material to dig, thus facilitating burrowing by gastropods [Consistent with other studies ,37,38, wstropods .C. aspersum, may act as opportunistic, shallow-burrowers rather than deep-burrowers before entering hibernation. This is in contrast with the behavior of other edible land snails of commercial importance. For example, the green snail, Helix aperta , can burrow itself into the ground as deep as 15 cm before overwintering [Helix lucorum  or Helix pomatia  [However, the burrowing snails did not migrate deep into the substratum, only burrowing down to 5 cm. The burrowing depth hence appears to be independent of the influence of soil texture. These data suggest that mature snails, intering ,41. Thiss, 1758) ,43.C. aspersum in farms using the ISISF system [Lutrasil frost cloth (LFC) is dense enough to keep warmth inside and has a microporous structure allowing rain to pass through and reach the soil . DesigneF system . All proF system . Such evF system . On one F system , thus afF system ,37, makiF system ,8. MoreoF system  and loweF system ,47.The use of HDPE instead of LFC as the upper layers for structures S3 and S4 was associated with lower amounts of moist straw thanfor structure S2. HDPE is known to block oxygen transfer and promote water condensation on the inside surfaces when applied tothe soil . Longer The thermal performance that a greenhouse provides overwinter is directly associated with the size of the covered area . This maC. aspersum, successfully passing the winter. Moreover, this death rate is below the normal range of mortalities in areas with milder winters, such as France, Turkey, or Southern California, whichis ~50–80% [Compelling evidence points to the survival of mature specimens during winter acting as a crucial factor in the population dynamics of both wild gastropod populations ,32,49,50 ~50–80% ,37,51.Helix pomatia, the brown garden snail, C. aspersum, has a limited ability to cool and survive ice formation in its tissues, being considered a partially freezing tolerant species. This gastropod can survive to below-zero temperatures for a relatively short period, amaximumof 9.8 h for exposure to temperatures as low as −5 °C [C. aspersum in Romania despite its success in colonizing new habitats [C. aspersum spread in a continuous areal in CentralEastern Europe.Unlike the native Roman snail as −5 °C . To the habitats . In facthabitats ,54; in Hhabitats . Moreovehabitats . Taken tMost shells of dead individuals were intact, and therefore, the potential causes of death may only be guessed: diseases, parasites, cold weather, premature arousal, starvation, and physical stress may all have been involved. Given the comparable distributions of this shell condition class across the pen(s) protected with and without the “sandwich” system, one can infer that cold weather may be, to some extent, responsible for the observed mortalities. The frequencies of smashed shells and shells with large holes were within a close range for all pens, lending support for rodents  as the main biotic factor affecting overwinter survival in investigated farms. This is consistent with data from other studies ,37. HoweC. aspersum juveniles, especially in Similar differences in entering and exit time, as well as the shorter duration of hibernation of juvenile snails, have been reported for wild populations of several snail species, which undergo winter hibernation, such as Helix lucorum  [Cepaea vindobonensis  [Juvenile snails with shell diameters less than 1 cm tended to enter later and awake earlier from hibernation than their mature counterparts. Indeed, individuals of similar age and size are known to display this habit and be active throughout the warmer winter periods . This bes, 1758)  and Cepac, 1821) .We also note that the length of hibernation was more than five months. This duration is close to those reported for the Atlantic coast of France, longer thanthose observed in Spain, but shorter than those identified in Scotland or Wales . Under tAlthough the burrowing behavior was not uncommon, most snails tended to hibernate clustered together, primarily attached to the lower surface of micro shelters and secondarily at the soil surface. When the protection system was dismantled, the highest survival tended to occur for individuals hibernating in large aggregations. This association between higher survival and overwintering gregariousness could indicate that these specimens have found particularly sheltered locations to take refuge during winter, where they may be protected against the direct impact of temperature, wind, or humidity . Snail aC. aspersum snails in colder climates.Mature gastropods in preparation for hibernation exhibited a significantly higher preference for wood and ridge-tile micro shelters than for corrugated iron micro shelters. Soil texture significantly influenced the burrowing behavior, but not the burrowing depth. All pilot structures tested displayed significantly higher thermal protection efficiency compared to the sole use of LFC. The balance between straw moistening and thermal protection favored using structure soil/LFC/straw/10-cm air cushion/HDPE—the “sandwich” system. Under farm conditions, snails tended to hibernate clustered together, attached to the lower surface of micro shelters. The use ofthe “sandwich” system yielded significantly higher thermal protection efficiency and snail survival thanthe sole use of LFC. Predator occurrence appeared to affect overwintering survival only marginally. These data suggest that the combined use of the “sandwich” system and ridge-tile/wood micro shelters could be an effective solution for overwintering mature